Etude de la régulation de l'autophagie au cours de la différenciation des cellules de leucémie aiguë promyélocytaire (rôles dans la survie et la différenciation cellulaire)

L autophagie, processus catabolique lysosomal de recyclage de constituants cellulaires, est essentielle à la survie, à la différenciation et au maintien de l homéostasie cellulaire. Ce processus est fréquemment impliqué dans la survie et la chimiorésistance des tumeurs. La leucémie aiguë promyélocytaire (LAP) est caractérisée par un blocage de la différenciation de la lignée hématopoïétique au stade promyélocytaire. Le traitement des LAP a considérablement progressé depuis l administration aux patients de doses pharmacologiques d acide rétinoïque tout-trans (ATRA), un puissant agent de différenciation. L objectif de ma thèse a consisté à étudier la régulation de l autophagie au cours de l induction de différenciation des cellules de LAP par l ATRA et de rechercher son implication éventuelle dans les mécanismes d action de ce traitement et les modes d échappement observés. Lors de mon travail de thèse, j ai mis en évidence une activation de l autophagie lors de l induction de la différenciation granulocytaire des cellules de LAP par l ATRA. J ai montré que cette réponse était associée à une inhibition de la voie mTOR et une induction de l expression des protéines BECLIN 1 et p62/SQSTM1. De façon intéressante, les cellules de LAP résistantes à la maturation par l ATRA ne sont pas capables d induire l expression de p62/SQSTM1 en réponse à l ATRA. De même, l expression de p62/SQSTM1 dans les blastes des patients atteints de leucémie aiguë myéloïde est plus faible que celle des granulocytes de sujets sains. L ensemble de ces données indique que l expression de p62/SQSTM1 est réprimée dans les phénotypes immatures des cellules myéloïdes mais au contraire induite dans les cellules leucémiques qui s engagent vers une différenciation terminale (granulocytes/neutrophiles). Enfin, j ai démontré que les protéines BECLIN 1 et p62/SQSTM1 sont essentielles à la survie de cellules de LAP matures mais non pas à l engagement de ces cellules vers la différenciation granulocytaire. Ainsi, ces résultats suggèrent qu en permettant la survie des cellules de LAP différenciées, p62/SQSTM1 et BECLIN 1 pourraient contribuer au développement des résistances à l ATRA et/ou à l induction des complications associées à ce traitement tel que le syndrome de différenciation.Autophagy, a lysosomal process used by the cell to degrade and recycle cytoplasmic constituents, is essential for cell survival, differentiation and the maintenance of cellular homeostasis. Autophagy is often involved in cell survival and resistance to anti-tumor therapy. Acute promyelocytic leukemia (APL) results from a blockade of granulocyte differentiation at the promyelocytic stage. All-trans retinoic acid (ATRA), a potent differentiation agent, has been shown to induce clinical remission in APL patients. The aim of our study was to investigate the regulation and roles of autophagy during ATRA-induced APL cells maturation into neutrophils/granulocytes with the ultimate objective to identify critical mechanisms involved in chemoresistance of APL patients. During my thesis, I demonstrated that autophagy is upregulated during the course of ATRA-induced neutrophil/granulocyte differentiation of APL cells. This response is associated with inhibition of mTOR activity and upregulation of both BECLIN 1 and p62/SQSTM1 proteins. Interestingly, induction of p62/SQSTM1 by ATRA was impaired in maturation-resistant NB4 cells but is re-activated when differentiation was restored in these cells. Accordingly, primary blast cells of AML patients exhibited significantly lower p62/SQSTM1 mRNA levels than did granulocytes from healthy donors. Together, these results highlight that p62/SQSTM1 expression level is repressed in immature myeloid cells compared to mature ones. Moreover, I demonstrated that BECLIN 1 and p62/SQSTM1 proteins are essential for the survival of myeloid cells that undergo differentiation but have no crucial effect on the granulocytic differentiation. This finding may help to elucidate the mechanisms involved in ATRA resistance of APL patients, and in the ATRA syndrome caused by an accumulation of mature APL cells.BORDEAUX2-Bib. électronique (335229905) / SudocBORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

The transglutaminase type 2 and pyruvate kinase isoenzyme M2 interplay in autophagy regulation

Autophagy is a self-degradative physiological process by which the cell removes worn-out or damaged components. Constant at basal level it may become highly active in response to cellular stress. The type 2 transglutaminase (TG2), which accumulates under stressful cell conditions, plays an important role in the regulation of autophagy and cells lacking this enzyme display impaired autophagy/mitophagy and a consequent shift their metabolism to glycolysis. To further define the molecular partners of TG2 involved in these cellular processes, we analysed the TG2 interactome under normal and starved conditions discovering that TG2 interacts with various proteins belonging to different functional categories. Herein we show that TG2 interacts with pyruvate kinase M2 (PKM2), a rate limiting enzyme of glycolysis which is responsible for maintaining a glycolytic phenotype in malignant cells and displays non metabolic functions, including transcriptional co-activation and protein kinase activity. Interestingly, the ablation of PKM2 led to the decrease of intracellular TG2's transamidating activity paralleled by an increase of its tyrosine phosphorylation. Along with this, a significant decrease of ULK1 and Beclin1 was also recorded, thus suggesting a block in the upstream regulation of autophagosome formation. These data suggest that the PKM2/TG2 interplay plays an important role in the regulation of autophagy in particular under cellular stressful conditions such as those displayed by cancer cells

Chikungunya virus-induced autophagy delays caspase-dependent cell death

Autophagy is an important survival pathway and can participate in the host response to infection. Studying Chikungunya virus (CHIKV), the causative agent of a major epidemic in India, Southeast Asia, and southern Europe, we reveal a novel mechanism by which autophagy limits cell death and mortality after infection. We use biochemical studies and single cell multispectral assays to demonstrate that direct infection triggers both apoptosis and autophagy. CHIKV-induced autophagy is mediated by the independent induction of endoplasmic reticulum and oxidative stress pathways. These cellular responses delay apoptotic cell death by inducing the IRE1α–XBP-1 pathway in conjunction with ROS-mediated mTOR inhibition. Silencing of autophagy genes resulted in enhanced intrinsic and extrinsic apoptosis, favoring viral propagation in cultured cells. Providing in vivo evidence for the relevance of our findings, Atg16L(HM) mice, which display reduced levels of autophagy, exhibited increased lethality and showed a higher sensitivity to CHIKV-induced apoptosis. Based on kinetic studies and the observation that features of apoptosis and autophagy were mutually exclusive, we conclude that autophagy inhibits caspase-dependent cell death but is ultimately overwhelmed by viral replication. Our study suggests that inducers of autophagy may limit the pathogenesis of acute Chikungunya disease

Exome sequencing of a colorectal cancer family reveals shared mutation pattern and predisposition circuitry along tumor pathways

The molecular basis of cancer and cancer multiple phenotypes are not yet fully understood. Next Generation Sequencing promises new insight into the role of genetic interactions in shaping the complexity of cancer. Aiming to outline the differences in mutation patterns between familial colorectal cancer cases and controls we analyzed whole exomes of cancer tissues and control samples from an extended colorectal cancer pedigree, providing one of the first data sets of exome sequencing of cancer in an African population against a background of large effective size typically with excess of variants. Tumors showed hMSH2 loss of function SNV consistent with Lynch syndrome. Sets of genes harboring insertions-deletions in tumor tissues revealed, however, significant GO enrichment, a feature that was not seen in control samples, suggesting that ordered insertions-deletions are central to tumorigenesis in this type of cancer. Network analysis identified multiple hub genes of centrality. ELAVL1/HuR showed remarkable centrality, interacting specially with genes harboring non-synonymous SNVs thus reinforcing the proposition of targeted mutagenesis in cancer pathways. A likely explanation to such mutation pattern is DNA/RNA editing, suggested here by nucleotide transition-to-transversion ratio that significantly departed from expected values (p-value 5e-6). NFKB1 also showed significant centrality along with ELAVL1, raising the suspicion of viral etiology given the known interaction between oncogenic viruses and these proteins

Modulation of the ATM/autophagy pathway by a G-quadruplex ligand tips the balance between senescence and apoptosis in cancer cells

G-quadruplex ligands exert their antiproliferative effects through telomere-dependent and telomere-independent mechanisms, but the inter-relationships among autophagy, cell growth arrest and cell death induced by these ligands remain largely unexplored. Here, we demonstrate that the G-quadruplex ligand 20A causes growth arrest of cancer cells in culture and in a HeLa cell xenografted mouse model. This response is associated with the induction of senescence and apoptosis. Transcriptomic analysis of 20A treated cells reveals a significant functional enrichment of biological pathways related to growth arrest, DNA damage response and the lysosomal pathway. 20A elicits global DNA damage but not telomeric damage and activates the ATM and autophagy pathways. Loss of ATM following 20A treatment inhibits both autophagy and senescence and sensitizes cells to death. Moreover, disruption of autophagy by deletion of two essential autophagy genes ATG5 and ATG7 leads to failure of CHK1 activation by 20A and subsequently increased cell death. Our results, therefore, identify the activation of ATM by 20A as a critical player in the balance between senescence and apoptosis and autophagy as one of the key mediators of such regulation. Thus, targeting the ATM/autophagy pathway might be a promising strategy to achieve the maximal anticancer effect of this compound

Simultaneous Induction of Non-Canonical Autophagy and Apoptosis in Cancer Cells by ROS-Dependent ERK and JNK Activation

Background: Chemotherapy-induced reduction in tumor load is a function of apoptotic cell death, orchestrated by intracellular caspases. However, the effectiveness of these therapies is compromised by mutations affecting specific genes, controlling and/or regulating apoptotic signaling. Therefore, it is desirable to identify novel pathways of cell death, which could function in tandem with or in the absence of efficient apoptotic machinery. In this regard, recent evidence supports the existence of a novel cell death pathway termed autophagy, which is activated upon growth factor deprivation or exposure to genotoxic compounds. The functional relevance of this pathway in terms of its ability to serve as a stress response or a truly death effector mechanism is still in question; however, reports indicate that autophagy is a specialized form of cell death under certain conditions. Methodology/Principal Findings: We report here the simultaneous induction of non-canonical autophagy and apoptosis in human cancer cells upon exposure to a small molecule compound that triggers intracellular hydrogen peroxide (H2O2) production. Whereas, silencing of beclin1 neither inhibited the hallmarks of autophagy nor the induction of cell death, Atg 7 or Ulk1 knockdown significantly abrogated drug-induced H2O2-mediated autophagy. Furthermore, we provide evidence that activated extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) are upstream effectors controlling both autophagy and apoptosis in response to elevated intracellular H2O2. Interestingly, inhibition of JNK activity reversed the increase in Atg7 expression in this system, thus indicating that JNK may regulate autophagy by activating Atg7. Of note, the small molecule compound triggered autophagy and apoptosis in primary cells derived from patients with lymphoma, but not in non-transformed cells. Conclusions/Significance: Considering that loss of tumor suppressor beclin 1 is associated with neoplasia, the ability of this small molecule compound to engage both autophagic and apoptotic machineries via ROS production and subsequent activation of ERK and JNK could have potential translational implications.Singapore. Biomedical Research CouncilSingapore. Ministry of Educatio

Cells

Stem cells isolated from the apical papilla of wisdom teeth (SCAPs) are an attractive model for tissue repair due to their availability, high proliferation rate and potential to differentiate in vitro towards mesodermal and neurogenic lineages. Adult stem cells, such as SCAPs, develop in stem cell niches in which the oxygen concentration [O] is low (3-8% compared with 21% of ambient air). In this work, we evaluate the impact of low [O] on the physiology of SCAPs isolated and processed in parallel at 21% or 3% O without any hyperoxic shock in ambient air during the experiment performed at 3% O. We demonstrate that SCAPs display a higher proliferation capacity at 3% O than in ambient air with elevated expression levels of two cell surface antigens: the alpha-6 integrin subunit (CD49f) and the embryonic stem cell marker (SSEA4). We show that the mesodermal differentiation potential of SCAPs is conserved at early passage in both [O], but is partly lost at late passage and low [O], conditions in which SCAPs proliferate efficiently without any sign of apoptosis. Unexpectedly, we show that autophagic flux is active in SCAPs irrespective of [O] and that this process remains high in cells even after prolonged exposure to 3% O

TBK1 Kinase Addiction in Lung Cancer Cells Is Mediated via Autophagy of Tax1bp1/Ndp52 and Non-Canonical NF-kappa B Signalling

K-Ras dependent non-small cell lung cancer (NSCLC) cells are 'addicted' to basal autophagy that reprograms cellular metabolism in a lysosomal-sensitive manner. Here we demonstrate that the xenophagy-associated kinase TBK1 drives basal autophagy, consistent with its known requirement in K-Ras-dependent NSCLC proliferation. Furthermore, basal autophagy in this context is characterised by sequestration of the xenophagy cargo receptor Ndp52 and its paralogue Tax1bp1, which we demonstrate here to be a bona fide cargo receptor. Autophagy of these cargo receptors promotes non-canonical NF-κB signalling. We propose that this TBK1-dependent mechanism for NF-κB signalling contributes to autophagy addiction in K-Ras driven NSCLC

The effects of baicalein and baicalin on mitochondrial function and dynamics: A review

Mitochondria play an essential role in cell survival by providing energy, calcium buffering, and regulating apoptosis. A growing body of evidence shows that mitochondrial dysfunction and its consequences, including impairment of the mitochondrial respiratory chain, excessive generation of reactive oxygen species, and excitotoxicity, play a pivotal role in the pathogenesis of different diseases such as neurodegenerative diseases, neuropsychiatric disorders, and cancer. The therapeutical role of flavonoids on these diseases is gaining increasing acceptance. Numerous studies on experimental models have revealed the favorable role of flavonoids on mitochondrial function and structure. This review highlights the promising role of baicalin and its aglycone form, baicalein, on mitochondrial function and structure with a focus on its therapeutic effects. We also discuss their chemistry, sources and bioavailability