The stress sensor GCN2 differentially controls ribosome biogenesis in colon cancer according to the nutritional context.

Nutrient availability is a key determinant of tumor cell behavior. While nutrient-rich conditions favor proliferation and tumor growth, scarcity, and particularly glutamine starvation, promotes cell dedifferentiation and chemoresistance. Here, linking ribosome biogenesis plasticity with tumor cell fate, we uncover that the amino acid sensor general control non-derepressible 2 (GCN2; also known as eIF-2-alpha kinase 4) represses the expression of the precursor of ribosomal RNA (rRNA), 47S, under metabolic stress. We show that blockade of GCN2 triggers cell death by an irremediable nucleolar stress and subsequent TP53-mediated apoptosis in patient-derived models of colon adenocarcinoma (COAD). In nutrient-rich conditions, a cell-autonomous GCN2 activity supports cell proliferation by stimulating 47S rRNA transcription, independently of the canonical integrated stress response (ISR) axis. Impairment of GCN2 activity prevents nuclear translocation of methionyl-tRNA synthetase (MetRS), resulting in nucleolar stress, mTORC1 inhibition and, ultimately, autophagy induction. Inhibition of the GCN2-MetRS axis drastically improves the cytotoxicity of RNA polymerase I (RNA pol I) inhibitors, including the first-line chemotherapy oxaliplatin, on patient-derived COAD tumoroids. Our data thus reveal that GCN2 differentially controls ribosome biogenesis according to the nutritional context. Furthermore, pharmacological co-inhibition of the two GCN2 branches and RNA pol I activity may represent a valuable strategy for elimination of proliferative and metabolically stressed COAD cells.

The hexosamine pathway and coat complex II promote malignant adaptation to nutrient scarcity.

The glucose-requiring hexosamine biosynthetic pathway (HBP), which produces UDP-N-acetylglucosamine for glycosylation reactions, promotes lung adenocarcinoma (LUAD) progression. However, lung tumor cells often reside in low-nutrient microenvironments, and whether the HBP is involved in the adaptation of LUAD to nutrient stress is unknown. Here, we show that the HBP and the coat complex II (COPII) play a key role in cell survival during glucose shortage. HBP up-regulation withstood low glucose-induced production of proteins bearing truncated N-glycans, in the endoplasmic reticulum. This function for the HBP, alongside COPII up-regulation, rescued cell surface expression of a subset of glycoproteins. Those included the epidermal growth factor receptor (EGFR), allowing an EGFR-dependent cell survival under low glucose in anchorage-independent growth. Accordingly, high expression of the HBP rate-limiting enzyme GFAT1 was associated with wild-type EGFR activation in LUAD patient samples. Notably, HBP and COPII up-regulation distinguished LUAD from the lung squamous-cell carcinoma subtype, thus uncovering adaptive mechanisms of LUAD to their harsh microenvironment.

Pemetrexed Hinders Translation Inhibition upon Low Glucose in Non-Small Cell Lung Cancer Cells.

Genetic alterations in non-small cell lung cancers (NSCLC) stimulate the generation of energy and biomass to promote tumor development. However, the efficacy of the translation process is finely regulated by stress sensors, themselves often controlled by nutrient availability and chemotoxic agents. Yet, the crosstalk between therapeutic treatment and glucose availability on cell mass generation remains understudied. Herein, we investigated the impact of pemetrexed (PEM) treatment, a first-line agent for NSCLC, on protein synthesis, depending on high or low glucose availability. PEM treatment drastically repressed cell mass and translation when glucose was abundant. Surprisingly, inhibition of protein synthesis caused by low glucose levels was partially dampened upon co-treatment with PEM. Moreover, PEM counteracted the elevation of the endoplasmic reticulum stress (ERS) signal produced upon low glucose availability, providing a molecular explanation for the differential impact of the drug on translation according to glucose levels. Collectively, these data indicate that the ERS constitutes a molecular crosstalk between microenvironmental stressors, contributing to translation reprogramming and proteostasis plasticity.

ATF4-Dependent NRF2 Transcriptional Regulation Promotes Antioxidant Protection during Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress generates reactive oxygen species (ROS) that induce apoptosis if left unabated. To limit oxidative insults, the ER stress PKR-like endoplasmic reticulum Kinase (PERK) has been reported to phosphorylate and activate nuclear factor erythroid 2-related factor 2 (NRF2). Here, we uncover an alternative mechanism for PERK-mediated NRF2 regulation in human cells that does not require direct phosphorylation. We show that the activation of the PERK pathway rapidly stimulates the expression of NRF2 through activating transcription factor 4 (ATF4). In addition, NRF2 activation is late and largely driven by reactive oxygen species (ROS) generated during late protein synthesis recovery, contributing to protecting against cell death. Thus, PERK-mediated NRF2 activation encompasses a PERK-ATF4-dependent control of NRF2 expression that contributes to the NRF2 protective response engaged during ER stress-induced ROS production.

Mesenchymal stem cells seeded on a human amniotic membrane improve liver regeneration and mouse survival after extended hepatectomy.

Liver failure remains the leading cause of post-operative mortality after hepatectomy. This study investigated the effect of treatment with allogenic mesenchymal stem cells (MSCs) on survival and liver regeneration 48 hr and 7 days after 80% hepatectomy in C57Bl/6 mice. To optimize their biodistribution, MSCs were grown on acellular human amniotic membranes (HAM) and applied as a patch on the remnant liver. This approach was compared with MSC infusion and HAM patch alone. Hepatectomized mice without any treatment were used as control group. Survival rate was calculated and biological and histopathological parameters were analysed to monitor liver function and regeneration. MSCs grown on HAM retained their ability to proliferate, to differentiate into osteoblasts and adipocytes and to respond to pro-inflammatory stimuli. Extended hepatectomy (80%) led to liver failure that resulted in death within 72 hr in 76% of mice. MSC infusion showed an early but transitory positive effect on survival. MSC/HAM patches stimulated regeneration and significantly improved survival rate (54% vs. 24% in the control group at 7 days). They also decreased the severity of hepatectomy-induced steatosis, suggesting a modulation of lipid metabolism in hepatocytes. MSCs were still present on HAM at Days 2 and 7 posthepatectomy. In conclusion, engineered tissue constructs that combine MSCs and HAM improve survival and liver regeneration after 80% hepatectomy in mice. These encouraging results pave the way to potential clinical application.

Epidermal Growth Factor Represses Constitutive Androstane Receptor Expression in Primary Human Hepatocytes and Favors Regulation by Pregnane X Receptor.

Growth factors have key roles in liver physiology and pathology, particularly by promoting cell proliferation and growth. Recently, it has been shown that in mouse hepatocytes, epidermal growth factor receptor (EGFR) plays a crucial role in the activation of the xenosensor constitutive androstane receptor (CAR) by the antiepileptic drug phenobarbital. Due to the species selectivity of CAR signaling, here we investigated epidermal growth factor (EGF) role in CAR signaling in primary human hepatocytes. Primary human hepatocytes were incubated with CITCO, a human CAR agonist, or with phenobarbital, an indirect CAR activator, in the presence or absence of EGF. CAR-dependent gene expression modulation and PXR involvement in these responses were assessed upon siRNA-based silencing of the genes that encode CAR and PXR. EGF significantly reduced CAR expression and prevented gene induction by CITCO and, to a lower extent, by phenobarbital. In the absence of EGF, phenobarbital and CITCO modulated the expression of 144 and 111 genes, respectively, in primary human hepatocytes. Among these genes, only 15 were regulated by CITCO and one by phenobarbital in a CAR-dependent manner. Conversely, in the presence of EGF, CITCO and phenobarbital modulated gene expression only in a CAR-independent and PXR-dependent manner. Overall, our findings suggest that in primary human hepatocytes, EGF suppresses specifically CAR signaling mainly through transcriptional regulation and drives the xenobiotic response toward a pregnane X receptor (PXR)-mediated mechanism.

Analysis of Glycogen Synthase Kinase Inhibitors That Regulate Cytochrome P450 Expression in Primary Human Hepatocytes by Activation of ß-Catenin, Aryl Hydrocarbon Receptor and Pregnane X Receptor Signaling.

Cytochrome P450 (CYP) expression and activity are not homogeneous in the liver lobules. Indeed, CYPs are mainly expressed and induced in centrilobular hepatocytes. The wingless-type MMTV integration site family (WNT)/ß-catenin pathway was identified as a major regulator of this zonal organization. We have recently demonstrated that in primary human hepatocytes (PHHs), the expression of CYP2E1, CYP1A2, and aryl hydrocarbon receptor (AhR), but not of CYP3A4, is regulated by the WNT/ß-catenin pathway in response to WNT3a, its canonical activator. Here, we investigated whether glycogen synthase kinase 3ß (GSK3ß) inhibitors, which mimic the action of WNT molecules, could be used in PHHs to activate the ß-catenin pathway to study CYP expression. We assessed the activity of 6BIO (6-bromoindirubin-3'-oxime), CHIR99021 (6-((2-((4-(2,4-dichlorophenyl)-5-(4methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino) nicotinonitrile), and GSK3iXV (Pyridocarbazolo-cyclopentadienyl Ruthenium complex GSK3 inhibitor XV) that belong to structurally different families of GSK3ß inhibitors. Using small interfering RNAs, reporter gene assays, and molecular docking predictions, we demonstrated that GSK3ß inhibitors can activate the WNT/ß-catenin pathway in PHHs to regulate CYP2E1 expression. We also found that 6BIO and GSK3iXV are AhR full agonists that participate, through AhR signaling, to CYP1A2 induction. Conversely, CHIR99021 is an AhR partial agonist, and a pregnane X receptor ligand and partial agonist, thus regulating CYP1A2 and CYP3A4 gene expression in a ß-catenin-independent manner. In conclusion, GSK3ß inhibitors can activate the WNT/ß-catenin pathway in PHHs. Nevertheless, their role in CYP regulation should be analyzed with caution as these molecules can interact with xenosensors.

Cold Preservation of Human Adult Hepatocytes for Liver Cell Therapy.

Hepatocyte transplantation is a promising alternative therapy for the treatment of hepatic failure, hepatocellular deficiency, and genetic metabolic disorders. Hypothermic preservation of isolated human hepatocytes is potentially a simple and convenient strategy to provide on-demand hepatocytes in sufficient quantity and of the quality required for biotherapy. In this study, first we assessed how cold storage in three clinically safe preservative solutions (UW, HTS-FRS, and IGL-1) affects the viability and in vitro functionality of human hepatocytes. Then we evaluated whether such cold-preserved human hepatocytes could engraft and repopulate damaged livers in a mouse model of liver failure. Human hepatocytes showed comparable viabilities after cold preservation in the three solutions. The ability of fresh and cold-stored hepatocytes to attach to a collagen substratum and to synthesize and secrete albumin, coagulation factor VII, and urea in the medium after 3 days in culture was also equally preserved. Cold-stored hepatocytes were then transplanted in the spleen of immunodeficient mice previously infected with adenoviruses containing a thymidine kinase construct and treated with a single dose of ganciclovir to induce liver injury. Engraftment and liver repopulation were monitored over time by measuring the blood level of human albumin and by assessing the expression of specific human hepatic mRNAs and proteins in the recipient livers by RT-PCR and immunohistochemistry, respectively. Our findings show that cold-stored human hepatocytes in IGL-1 and HTS-FRS preservative solutions can survive, engraft, and proliferate in a damaged mouse liver. These results demonstrate the usefulness of human hepatocyte hypothermic preservation for cell transplantation.

The WNT/ß-catenin pathway is a transcriptional regulator of CYP2E1, CYP1A2, and aryl hydrocarbon receptor gene expression in primary human hepatocytes.

The wingless-type MMTV integration site family (WNT)/ß-catenin/adenomatous polyposis coli (CTNNB1/APC) pathway has been identified as a regulator of drug-metabolizing enzymes in the rodent liver. Conversely, little is known about the role of this pathway in drug metabolism regulation in human liver. Primary human hepatocytes (PHHs), which are the most physiologically relevant culture system to study drug metabolism in vitro, were used to investigate this issue. This study assessed the link between cytochrome P450 expression and WNT/ß-catenin pathway activity in PHHs by modulating its activity with recombinant mouse Wnt3a (the canonical activator), inhibitors of glycogen synthase kinase 3ß, and small-interfering RNA to invalidate CTNNB1 or its repressor APC, used separately or in combination. We found that the WNT/ß-catenin pathway can be activated in PHHs, as assessed by universal ß-catenin target gene expression, leucine-rich repeat containing G protein-coupled receptor 5. Moreover, WNT/ß-catenin pathway activation induces the expression of CYP2E1, CYP1A2, and aryl hydrocarbon receptor, but not of CYP3A4, hepatocyte nuclear factor-4α, or pregnane X receptor (PXR) in PHHs. Specifically, we show for the first time that CYP2E1 is transcriptionally regulated by the WNT/ß-catenin pathway. Moreover, CYP2E1 induction was accompanied by an increase in its metabolic activity, as indicated by the increased production of 6-OH-chlorzoxazone and by glutathione depletion after incubation with high doses of acetaminophen. In conclusion, the WNT/ß-catenin pathway is functional in PHHs, and its induction in PHHs represents a powerful tool to evaluate the hepatotoxicity of drugs that are metabolized by CYP2E1.

Usage of adenovirus expressing thymidine kinase mediated hepatocellular damage for enabling mouse liver repopulation with allogenic or xenogenic hepatocytes.

It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2(-/-) γc(-/-) mice using an adenovirus encoding herpes virus thymidine kinase (AdTk) and two consecutive doses of ganciclovir (GCV). We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line.

Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells.

Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56(+) fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non-human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H(2) O(2) )-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage.

Isolation and culture of adult human liver progenitor cells: in vitro differentiation to hepatocyte-like cells.

Highly differentiated normal human hepatocytes represent the gold standard cellular model for basic and applied research in liver physiopathology, pharmacology, toxicology, virology, and liver biotherapy. Nowadays, although livers from organ donors or medically required resections represent the current sources of hepatocytes, the possibility to generate hepatocytes from the differentiation of adult and embryonic stem cells represents a promising opportunity. The aim of this chapter is to describe our experience with the isolation from adult human liver and culture of non-parenchymal epithelial cells. Under appropriate conditions, these cells differentiate in vitro in hepatocyte-like cells and therefore appear to behave as liver progenitor cells.

Isolation, characterization, and differentiation to hepatocyte-like cells of nonparenchymal epithelial cells from adult human liver.

Activation and proliferation of human liver progenitor cells has been observed during acute and chronic liver diseases. Our goal was to investigate the presence of these putative progenitors in the liver of patients who underwent lobectomy for various reasons but did not show any hepatic insufficiency. Hepatic lesions were evaluated by histological analysis. Nonparenchymal epithelial (NPE) cells were isolated from samples of human liver resections located at a distance from the lesion that motivated the operation and were cultured and characterized. These cells exhibited a marked proliferative potential. They did not express the classic set of stem cell/progenitor markers (Oct-4, Rex-1, alpha-fetoprotein, CD90, c-kit, and CD34) and were faintly positive for albumin. When cultured at confluence in the presence of hepatocyte growth factor and either epidermal growth factor or fibroblast growth factor-4, they entered a differentiation process toward hepatocytes. Their phenotype was quantitatively compared with that of mature human hepatocytes in primary culture. Differentiated NPE cells expressed albumin; alpha1-antitrypsin; fibrinogen; hepatobiliary markers such as cytokeratins 7, 19, and 8/18; liver-enriched transcription factors; and genes characterized by either a fetal (cytochrome P4503A7 and glutathione S-transferase pi) or a mature (tyrosine aminotransferase, tryptophan 2,3-dioxygenase, glutathione S-transferase alpha, and cytochrome P4503A4) expression pattern. NPE cells could be isolated from the liver of several patients, irrespective of the absence or presence of lesions, and differentiated toward hepatocyte-like cells with an intermediate hepatobiliary and mature/immature phenotype. These cells are likely to represent a resident progenitor population of the adult human liver, even in the absence of hepatic failure. Disclosure of potential conflicts of interest is found at the end of this article.

Ketoconazole and miconazole are antagonists of the human glucocorticoid receptor: consequences on the expression and function of the constitutive androstane receptor and the pregnane X receptor.

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) play a major part in the control of drug metabolism and transport. We have previously shown that PXR and CAR expression is controlled by the glucocorticoid receptor (GR) and proposed the existence of a signal transmission cascade GR-(PXR/CAR)-drug metabolizing and transporter systems. In the current study, we investigated the effect of ketoconazole and other azole-derived drugs, miconazole and fluconazole, on the transcriptional activity of the human GR (hGR) in HeLa and HepG2 cells, and in primary human hepatocytes. The data show that ketoconazole inhibits GR transcriptional activity and competes with dexamethasone for hGR binding. In primary human hepatocytes, ketoconazole inhibits the expression of 1) GR-responsive genes tyrosine aminotransferase and both PXR and CAR; 2) CAR and PXR target genes, including cytochromes P450 (P450) CYP2B6, CYP2C9, and CYP3A4; UDP-glucuronosyltransferase 1A1, glutathione S-transferases A1 and A2; and transporter proteins (phase III) solute carrier family 21 form A6 and multidrug resistance protein 2. In parallel experiments, ketoconazole affected neither the expression of GR, the expression of glyceraldehyde-3-phosphate dehydrogenase, nor the inducible expression of CYP1A1 and 1A2. Miconazole behaved like ketoconazole, whereas fluconazole had no effect. We conclude that, in addition to their well known inhibitory effect on P450 enzyme activities, ketoconazole and miconazole are antagonists of hGR. These results provide a novel molecular mechanism by which these compounds may exert adverse and toxic effects on drug metabolism and other functions in human.