Fabienne Guillaumond

The circadian clock component BMAL1 is a critical regulator of p21WAF1/CIP1 expression and hepatocyte proliferation.

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.

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.

Kruppel-like factor KLF10 is a link between the circadian clock and metabolism in liver.

ABSTRACT 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.

How nuclear receptors tell time.

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.

Altered Stra13 and Dec2 circadian gene expression in hypoxic cells.

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.

Defects in mitophagy promote redox‐driven metabolic syndrome in the absence of TP53INP1

The metabolic syndrome covers metabolic abnormalities including obesity and type 2 diabetes (T2D). T2D is characterized by insulin resistance resulting from both environmental and genetic factors. A genome‐wide association study (GWAS) published in 2010 identified TP53INP1 as a new T2D susceptibility locus, but a pathological mechanism was not identified. In this work, we show that mice lacking TP53INP1 are prone to redox‐driven obesity and insulin resistance. Furthermore, we demonstrate that the reactive oxygen species increase in TP53INP1‐deficient cells results from accumulation of defective mitochondria associated with impaired PINK/PARKIN mitophagy. This chronic oxidative stress also favors accumulation of lipid droplets. Taken together, our data provide evidence that the GWAS‐identified TP53INP1 gene prevents metabolic syndrome, through a mechanism involving prevention of oxidative stress by mitochondrial homeostasis regulation. In conclusion, this study highlights TP53INP1 as a molecular regulator of redox‐driven metabolic syndrome and provides a new preclinical mouse model for metabolic syndrome clinical research.

LDL Receptor: An open route to feed pancreatic tumor cells

ABSTRACT The role of altered lipid metabolism in pancreatic ductal adenocarcinoma (PDAC) is poorly appreciated. We recently identified the lipid signature of PDAC and revealed low-density lipoprotein receptor (Ldlr) as a metabolic driver of this disease. Here, we comment our findings that disruption of Ldlr leads to intratumoral cholesterol imbalance and improves chemotherapy efficiency.

Influence of the Tumor Microenvironment on Cancer Cells Metabolic Reprogramming

As with castles, tumor cells are fortified by surrounding non-malignant cells, such as cancer-associated fibroblasts, immune cells, but also nerve fibers and extracellular matrix. In most cancers, this fortification creates a considerable solid pressure which limits oxygen and nutrient delivery to the tumor cells and causes a hypoxic and nutritional stress. Consequently, tumor cells have to adapt their metabolism to survive and proliferate in this harsh microenvironment. To satisfy their need in energy and biomass, tumor cells develop new capacities to benefit from metabolites of the microenvironment, either by their uptake through the macropinocytosis process or through metabolite transporters, or by a cross-talk with stromal cells and capture of extracellular vesicles that are released by the neighboring cells. However, the microenvironments of primary tumor and metastatic niches differ tremendously in their cellular/acellular components and available nutrients. Therefore, cancer cells must develop a metabolic flexibility conferring on them the ability to satisfy their biomass and energetic demands at both primary and metastasis sites. In this review, we propose a brief overview of how proliferating cancer cells take advantage of their surrounding microenvironment to satisfy their high metabolic demand at both primary and metastasis sites.

[Pancreatic adenocarcinoma: a tumor highly dependent on lipoproteins].

> 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.

Abstract 4396: Multiomics assessment of the cancer and stromal compartments of patient-derived pancreatic xenografts reveals clinically-relevant subtypes and novel targeted therapies

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, Aurelie 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, Aurelien de Reynies, 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