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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.

Summary Mitochondrial respiration (oxidative phosphorylation, OXPHOS) is an emerging target in currently refractory cancers such as pancreatic ductal adenocarcinoma (PDAC). However, the variability of energetic metabolic adaptations between PDAC patients has not been assessed in functional investigations. In this work, we demonstrate that OXPHOS rates are highly heterogeneous between patient tumors, and that high OXPHOS tumors are enriched in mitochondrial respiratory complex I at protein and mRNA levels. Therefore, we treated PDAC cells with phenformin (complex I inhibitor) in combination with standard chemotherapy (gemcitabine), showing that this treatment is synergistic specifically in high OXPHOS cells. Furthermore, phenformin cooperates with gemcitabine in high OXPHOS tumors in two orthotopic mouse models (xenografts and syngeneic allografts). In conclusion, this work proposes a strategy to identify PDAC patients likely to respond to the targeting of mitochondrial energetic metabolism in combination with chemotherapy, and that phenformin should be clinically tested in appropriate PDAC patient subpopulations., Graphical Abstract, Highlights Pancreatic ductal adenocarcinoma (PDAC) displays OXPHOS heterogeneity High OXPHOS PDAC tumors are enriched in mitochondrial respiratory complex I Complex I inhibitor phenformin synergizes with chemotherapy in high OXPHOS cells Phenformin cooperates with gemcitabine antitumoral activity in high OXPHOS tumors, Masoud et al. reveal that pancreatic cancer patients can be stratified according to their mitochondrial oxidative phosphorylation (OXPHOS) activity and to the expression of mitochondrial respiratory complex I. Targeting mitochondrial respiration with the complex I inhibitor phenformin cooperates with gemcitabine to eradicate high OXPHOS pancreatic cancer cells.

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.

Synthetic nucleoside analogues with novel structural entities are of considerable importance in the search for new structural paradigms with biologically interesting activities. We report in this work that microwave promoted C–O coupling reaction is able to efficiently offer a large array of novel O-arylated triazole nucleosides with considerably improved yields and appreciably reduced reaction time. One of the synthesized compounds showed interesting anticancer activity against human prostate and pancreatic cancers. The synthetic method described in this work not only highlights the importance of microwave irradiation in organic synthesis but also provides a promising access towards synthesizing novel nucleoside analogues which can offer molecular diversity in the quest for new and potential drug candidates.

International audience; Metabolic reprogramming is a hallmark of cancer development, mediated by genetic and epigenetic alterations that may be pharmacologically targeted. Among oncogenes, the kinase Akt is commonly overexpressed in tumors and favors glycolysis, providing a rationale for using Akt inhibitors. Here, we addressed the question of whether and how inhibiting Akt activity could improve therapy of non-small cell lung cancer (NSCLC) that represents more than 80% of all lung cancer cases. First, we demonstrated that Akt inhibitors interacted synergistically with Microtubule-Targeting Agents (MTAs) and specifically in cancer cell lines, including those resistant to chemotherapy agents and anti-EGFR targeted therapies. In vivo, we further revealed that the chronic administration of low-doses of paclitaxel - i.e. metronomic scheduling - and the anti-Akt perifosine was the most efficient and the best tolerated treatment against NSCLC. Regarding drug mechanism of action, perifosine potentiated the pro-apoptotic effects of paclitaxel, independently of cell cycle arrest, and combining paclitaxel/perifosine resulted in a sustained suppression of glycolytic and mitochondrial metabolism. This study points out that targeting cancer cell bioenergetics may represent a novel therapeutic avenue in NSCLC, and provides a strong foundation for future clinical trials of metronomic MTAs combined with Akt inhibitors.

Among the various directions explored in order to have a large number of differentiated articular chondrocytes easily available, the restoration of the differentiated properties after cell multiplication in monolayer has been proposed. It has been clearly shown that the synthesis of cartilage proteoglycans and type II collagen synthesis is coincident with the presence of a faint microfibrillar architecture but is absent in chondrocytes showing well-defined actin cables. Staurosporin, mainly described as a protein kinase C inhibitor, has also been shown to rapidly induce the disruption of the actin microfilaments. The purpose of this paper was to investigate whether properties of differentiated chondrocytes were reinitiated upon staurosporin treatment of serially passaged chondrocytes. Results showed, after staurosporine treatment of cells at Passage two for 5 d, complete suppression of type I and type III collagen synthesis and induction of type II collagen synthesis and of Alcian blue stainable matrix. Additionally, we showed that staurosporin restored metabolic responses that chondrocytes in primary culture exhibit upon interleukin-1 beta treatment (decrease of Alcian blue- positive cells, induction of expression of the 92 kDa gelatinase, nitric oxide production). We conclude that staurosporin is a potent redifferentiating agent of articular chondrocytes that have been subcultured up to Passage two for multiplication. Taking into account that the cellularity of cartilage is very low, staurosporine-treated chondrocytes could be useful as an alternative cellular model to evaluate pharmacotoxicological effects of drugs.

The use of Fluoresceincadaverine as a primary amine donor for detecting the endogenous substrates for active transglutaminase in living cells was studied. Fluoresceincadaverine was found to be suitable for labelling cells in culture as it did not induce cytotoxicity when used at 0.5 mM in culture media and diffused throughout the cell. After appropriate fixation using methanol, Fluoresceincadaverine-labelled cells were observed by direct fluorescence microscopy, allowing visualization of the substrates for active transglutaminase. Simultaneous detection of transglutaminase and of Fluoresceincadaverine incorporated into proteins strongly suggested that cytosolic transglutaminase was inactive in these living cells. However, transglutaminase co-distributed with Fluoresceincadaverine-labelled structures, which resembled a lattice. Fluoresceincadaverine-labelled proteins detected by Western blotting using an anti-Fluorescein antibody showed that, in living cells, the major transglutaminase substrate migrated at an apparent molecular weight of 220 kDa, as does fibronectin. Fibronectin was found to co-distribute with Fluoresceincadaverine-labelled lattice. This confirmed that these lattice structures were extracellular and, therefore, that transglutaminase is in an active form in this compartment. This opportunity to perform morphological and biochemical analyses in the search for transglutaminase substrates in living cells should help in determining the specific function of transglutaminases in a particular cell type as well as in universal cellular events, such as apoptosis or cell growth.

International audience; Background: The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT. Methodology/Principal Findings: In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC50) of 200 ± 40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC50 of 150 ± 80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant. Conclusions: Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity

In vivo, articular chondrocytes produce an important amount of extracellular matrix (cartilage) whose quality is impaired upon inflammation or aging leading to arthritis or arthrosis. Transglutaminases (EC 2.3.2.13) are a family of enzymes which have been shown to be involved in extracellular matrix stabilization, cell differentiation and possibly in initiation and propagation of inflammatory diseases. It is therefore of interest to study transglutaminase activity in chondrocytes. Transglutaminase activity was studied in rabbit articular chondrocytes in primary culture, where cells are in a well-differentiated state as assessed by collagen-type synthesis, as well as in subculture and in retinoic acid-treated cells, where cells are in a dedifferentiated state. Results showed that two different TGases activities are expressed in chondrocytes. One, down-regulated upon retinoic acid treatment of cells preferentially membrane bound and strongly activated upon trypsin treatment of cell lysates, is expressed at a high level in primary culture. The other one is up-regulated upon retinoic acid treatment, preferentially cytosolic and inactivated upon trypsin treatment of cell lysates. The rate of expression of the TGase down-regulated by RA seems to correlate with the differentiation state of the chondrocyte. This suggests that this TGase activity may have a physiological role in cartilage and merits further study.

Depending on the cell type studied, the involvement of type II transglutaminase (TGase) has been proposed in almost any event of the cell life such as differentiation, apoptosis, growth, aging, cell morphology and adhesion, metastatic capacity or extracellular matrix stabilization. In order to define the field(s) where this enzyme may be implicated in chondrocytes, type II TGase expression was studied in chondrocytes at different passages which differentiated state was modulated by retinoic acid, dihydrocytochalasin B or staurosporin. Results showed that (i) type II TGase expression is not incompatible with type II collagen expression, a main marker of chondrocyte differentiation (ii) type II TGase expression is higher when cells are in the exponential phase of growth than when growth arrested (iii) a high type II TGase expression does not imply that cells are apoptotic although cell apoptosis correlates with increased type II TGase expression (iv) non-adherent cells do not express type II TGase whereas adherent cells do whatever their differentiation state as assessed by type II collagen synthesis. These results suggest that, in articular chondrocytes, type II TGase is specifically implicated in the cell adhesion capacity.