Georges Stepien

The eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression

In response to different environmental stresses, eIF2α phosphorylation represses global translation coincident with preferential translation of ATF4, a master regulator controlling the transcription of key genes essential for adaptative functions. Here, we establish that the eIF2α/ATF4 pathway directs an autophagy gene transcriptional program in response to amino acid starvation or endoplasmic reticulum stress. The eIF2α-kinases GCN2 and PERK and the transcription factors ATF4 and CHOP are also required to increase the transcription of a set of genes implicated in the formation, elongation and function of the autophagosome. We also identify three classes of autophagy genes according to their dependence on ATF4 and CHOP and the binding of these factors to specific promoter cis elements. Furthermore, different combinations of CHOP and ATF4 bindings to target promoters allow the trigger of a differential transcriptional response according to the stress intensity. Overall, this study reveals a novel regulatory role of the eIF2α–ATF4 pathway in the fine-tuning of the autophagy gene transcription program in response to stresses.

Adenine nucleotide translocase 2 is a key mitochondrial protein in cancer metabolism

Adenine nucleotide translocase (ANT), a mitochondrial protein that facilitates the exchange of ADP and ATP across the mitochondrial inner membrane, plays an essential role in cellular energy metabolism. Human ANT presents four isoforms (ANT1-4), each with a specific expression depending on the nature of the tissue, cell type, developmental stage and status of cell proliferation. Thus, ANT1 is specific to muscle and brain tissues; ANT2 occurs mainly in proliferative, undifferentiated cells; ANT3 is ubiquitous; and ANT4 is found in germ cells. ANT1 and ANT3 export the ATP produced by oxidative phosphorylation (OxPhos) from the mitochondria into the cytosol while importing ADP. In contrast, the expression of ANT2, which is linked to the rate of glycolytic metabolism, is an important indicator of carcinogenesis. In fact, cancers are characterized by major metabolic changes that switch cells from the normally dual oxidative and glycolytic metabolisms to an almost exclusively glycolytic metabolism. When OxPhos activity is impaired, ANT2 imports glycolytically produced ATP into the mitochondria. In the mitochondrial matrix, the F1F0-ATPase complex hydrolyzes the ATP, pumping out a proton into the intermembrane space. The reverse operations of ANT2 and F1F0-ATPase under glycolytic conditions contribute to maintaining the mitochondrial membrane potential, ensuring cell survival and proliferation. Unlike the ANT1 and ANT3 isoforms, ANT2 is not pro-apoptotic and may therefore contribute to carcinogenesis. Since the expression of ANT2 is closely linked to the mitochondrial bioenergetics of tumors, it should be taken into account for individualizing cancer treatments and for the development of anticancer strategies.

Quantitative two-dimensional HRMAS 1H-NMR spectroscopy-based metabolite profiling of human cancer cell lines and response to chemotherapy.

NMR spectroscopy‐based metabolomics still needs development in quantification procedures. A method was designed for quantitative two‐dimensional high resolution magic angle spinning (HRMAS) proton‐NMR spectroscopy‐based metabolite profiling of intact cells. It uses referencing of metabolite‐related NMR signals to protein‐related NMR signals and yields straightforward and automatable metabolite profiling. The method enables exploitation of only two‐dimensionally visible metabolites and combination of one‐ and two‐dimensional spectra, thus providing an appreciable number of screened metabolites. With this procedure, 32 intracellular metabolites were attributed and quantified in human normal fibroblasts and tumor cells. The phenotype of several tumor cell lines (MCF7, PC3, 143B, and HepG2) was characterized by high levels of glutathione in cell lines with the higher proliferation rate, high levels of creatine, low levels of free amino acids, increased levels of phospholipid derivatives (mostly phosphocholine), and lower lactate content in cell lines with the higher proliferation rate. Other metabolites such as fatty acids differed widely among tumor cell lines. The response of tumor cell lines to chemotherapy also was evaluated by differential metabolite profiling, bringing insights into drug cytotoxicity and tumor cell adaptive mechanisms. The method may prove widely applicable to tumor cell phenotyping. Magn Reson Med 63:1172–1183, 2010.

Mitochondrial bioenergetic background confers a survival advantage to HepG2 cells in response to chemotherapy

Cancer cells mainly rely on glycolysis for energetic needs, and mitochondrial ATP production is almost inactive. However, cancer cells require the integrity of mitochondrial functions for their survival, such as the maintenance of the internal membrane potential gradient (ΔΨm). It thus may be predicted that ΔΨm regeneration should depend on cellular capability to produce sufficient ATP by upregulating glycolysis or recruiting oxidative phosphorylation (OXPHOS). To investigate this hypothesis, we compared the response to an anticancer agent chloroethylnitrosourea (CENU) of two transformed cell lines: HepG2 (hepatocarcinoma) with a partially differentiated phenotype and 143B (osteosarcoma) with an undifferentiated one. These cells types differ by their mitochondrial OXPHOS background; the most severely impaired being that of 143B cells. Treatment effects were tested on cell proliferation, O2 consumption/ATP production coupling, ΔΨm maintenance, and global metabolite profiling by NMR spectroscopy. Our results showed an OXPHOS uncoupling and a lowered ΔΨm, leading to an increased energy request to regenerate ΔΨm in both models. However, energy request could not be met by undifferentiated cells 143B, which ATP content decreased after 48 h leading to cell death, while partially differentiated cells (HepG2) could activate their oxidative metabolism and escape chemotherapy. We propose that mitochondrial OXPHOS background confers a survival advantage to more differentiated cells in response to chemotherapy. This suggests that the mitochondrial bioenergetic background of tumors should be considered for anticancer treatment personalization.

Combined methionine deprivation and chloroethylnitrosourea have time-dependent therapeutic synergy on melanoma tumors that NMR spectroscopy-based metabolomics explains by methionine and phospholipid metabolism reprogramming.

Methionine (Met) deprivation stress (MDS) is proposed in association with chemotherapy in the treatment of some cancers. A synergistic effect of this combination is generally acknowledged. However, little is known on the mechanism of the response to this therapeutic strategy. A model of B16 melanoma tumor in vivo was treated by MDS alone and in combination with chloroethylnitrosourea (CENU). It was applied recent developments in proton-NMR spectroscopy-based metabolomics for providing information on the metabolic response of tumors to MDS and combination with chemotherapy. MDS inhibited tumor growth during the deprivation period and growth resumption thereafter. The combination of MDS with CENU induced an effective time-dependent synergy on growth inhibition. Metabolite profiling during MDS showed a decreased Met content (P < 0.01) despite the preservation of the protein content, disorders in sulfur-containing amino acids, glutamine/proline, and phospholipid metabolism [increase of glycerophosphorylcholine (P < 0.01), decrease in phosphocholine (P < 0.05)]. The metabolic profile of MDS combined with CENU and ANOVA analysis revealed the implication of Met and phospholipid metabolism in the observed synergy, which may be interpreted as a Met–sparing metabolic reprogramming of tumors. It follows that combination therapy of MDS with CENU seems to intensify adaptive processes, which may set limitations to this therapeutic strategy.

Computational identification of transcriptionally co-regulated genes, validation with the four ANT isoform genes

BackgroundThe analysis of gene promoters is essential to understand the mechanisms of transcriptional regulation required under the effects of physiological processes, nutritional intake or pathologies. In higher eukaryotes, transcriptional regulation implies the recruitment of a set of regulatory proteins that bind on combinations of nucleotide motifs. We developed a computational analysis of promoter nucleotide sequences, to identify co-regulated genes by combining several programs that allowed us to build regulatory models and perform a crossed analysis on several databases. This strategy was tested on a set of four human genes encoding isoforms 1 to 4 of the mitochondrial ADP/ATP carrier ANT. Each isoform has a specific tissue expression profile linked to its role in cellular bioenergetics.ResultsFrom their promoter sequence and from the phylogenetic evolution of these ANT genes in mammals, we constructed combinations of specific regulatory elements. These models were screened using the full human genome and databases of promoter sequences from human and several other mammalian species. For each of transcriptionally regulated ANT1, 2 and 4 genes, a set of co-regulated genes was identified and their over-expression was verified in microarray databases.ConclusionsMost of the identified genes encode proteins with a cellular function and specificity in agreement with those of the corresponding ANT isoform. Our in silico study shows that the tissue specific gene expression is mainly driven by promoter regulatory sequences located up to about a thousand base pairs upstream the transcription start site. Moreover, this computational strategy on the study of regulatory pathways should provide, along with transcriptomics and metabolomics, data to construct cellular metabolic networks.

Computational analysis of the transcriptional regulation of the adenine nucleotide translocator isoform 4 gene and its role in spermatozoid glycolytic metabolism

Computational phylogenetic analysis coupled to promoter sequence alignment was used to understand mechanisms of transcriptional regulation and to identify potentially coregulated genes. Our strategy was validated on the human ANT4 gene which encodes the fourth isoform of the mitochondrial adenine nucleotide translocator specifically expressed during spermatogenesis. The movement of sperm flagella is driven mainly by ATP generated by glycolytic pathways, and the specific induction of the mitochondrial ANT4 protein presented an interesting puzzle. We analysed the sequences of the promoters, introns and exons of 30 mammalian ANT4 genes and constructed regulatory models. The whole human genome and promoter database were screened for genes that were potentially regulated by the generated models. 80% of the identified co-regulated genes encoded proteins with specific roles in spermatogenesis and with functions linked to male reproduction. Our in silico study enabled us to precise the specific role of the ANT4 isoform in spermatozoid bioenergetics.

A model of phospholipid biosynthesis in tumor in response to an anticancer agent in vivo.

We study, in this paper, a model for the core of the system of the Glycerophospholipid metabolism in the murine cells. It comprises the simple and enzymatic reactions of PhosphatidylEthanolamine and the PhosphatidylCholine. The models general structure is taken from a number of books and articles. We translate this model into a set of ordinary differential equations (ODEs), to propose a quantitative explanation of the experimental experiences and the observed results. In order to make it usable as a basis for simulations and mathematical analysis we need to make precise the various constants present in the equations but which are usually not directly accessible in the literature. In a first step we considered experimental data of rats liver cells obtained by NMR spectroscopy: given the values of metabolite concentrations we find appropriate parameter values which allow us to describe the system with ODEs. We have then performed several analyses using the developed model such as stability analysis. A first interesting result is the global stability of the system which was observed by simulation and then proved by mathematical arguments. A second important result is that we observe on the diagrams that the steady state for normal cells is precisely a singular point of order two, whereas tumoral cells present different characteristics; this fact has been proved for PhosphatidylEthanolamine N-Methyl transferase (PEMT), an enzyme which seems to be identified for the first time as a crucial element in the tumoral process. In a second step we applied our model to experimental data of proton HRMAS NMR spectroscopy for solid B16 melanoma and Lewis lung (3LL) 3LL carcinoma cells treated by Chloroethyl Nitrosourea (CENU). We performed a complete comparative analysis of parameters in order to learn the predictive statements to explain increases and decreases which one can observe in concentrations.

Abstract 62: Alteration of metabolic pathways of glucose consumption by CENU treatment in B16 melanoma tumors: A NMR spectroscopy-based [1,2-13C]glucose fluxomics analysis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnWe have previously shown using proton NMR spectroscopy-based metabolomics that mice bearing B16 melanoma tumors treated by chloroethylnitrosourea (CENU) had a strong intratumoral decrease in glycolysis involving the accumulation of intracellular glucose and glucose-6-phosphate, together the accumulation of glutamate derivatives, despite no significant lactate content change (1).nnTo get insights into the metabolic pathway followed by glucose carbons in CENU-treated tumor, we used double labelled glucose ([1,2-13C]glucose) and investigated the obtained isotopomers of lactate, the final product of glycolysis. Mice bearing B16 melanoma tumors were treated by CENU or saline solution (control) at days 11, 14 and 17 at dose 15µg/g weight. At day 23, mice were injected intraperitoneally with [1,2-13C]glucose at 25 mg in 150µl saline solution. Animals were sacrificed 30 minutes after glucose injection. Tumors were removed and kept at −80°C until use. The analysis of lactate isotopomers, a fluxomics approach, was done at 500 MHz using high resolution magic angle spinning (HRMAS) 1H-NMR spectroscopy by exploiting 1H-13C coupling signals. The two lactate signals, the methyl (C3) signal centered at 1.33 ppm and the methine (C2) signal centered at 4.12 ppm were differentially analyzed, and quantified from spectra witout and with broadband 13C-decoupling. In addition, the activity of pyruvate-kinase was measured using enzyme assay.nnIn comparison with control tumors, CENU-treated tumors exhibited an overall decrease of labelled lactate both at C3 and C2 position, together with a significant increase in the relative proportion of C2 to C3 labelling (0.65±0.10 vs 0.91±0.13, P<0.01, n=3 vs n=3). Because lactate only labelled on C3 was a product of glucose metabolism through the pentose phosphate pathway, it was concluded that most of glucose in CENU-treated tumors was directly metabolized to lactate, thus dedicated to ATP production rather than macromolecular biosynthesis. Consistently, the activity of pyruvate kinase, the main source of glycolysis-derived ATP was increased under CENU treatment (+48%, P<0.01).nnAltogether these fluxomics data provide evidence of glycolysis adaptation that may be correlated to reduced proliferation, decreased aggressiveness, and redifferentiation, as previously shown in this model (2). In addition, this study suggests that metabolic targeting of glycolysis-related ATP production may improve CENU efficacy.nn1- Morvan D, Demidem A. Cancer Res, 67; 2150-59, 2007nn2- Demidem A et al. Cancer Res, 61; 2294-300, 2001nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 62.