Séverine Lorin

Autophagy regulation and its role in cancer

The modulation of macroautophagy is now recognized as one of the hallmarks of cancer cells. There is accumulating evidence that autophagy plays a role in the various stages of tumorigenesis. Depending on the type of cancer and the context, macroautophagy can be tumor suppressor or it can help cancer cells to overcome metabolic stress and the cytotoxicity of chemotherapy. Recent studies have shed light on the role of macroautophagy in tumor-initiating cells, in tumor immune response cross-talk with the microenvironment. This review is intended to provide an up-date on these aspects, and to discuss them with regard to the role of the major signaling sub-networks involved in tumor progression (Beclin 1, MTOR, p53 and RAS) and in regulating autophagy.

Evidence for the interplay between JNK and p53-DRAM signaling pathways in the regulation of autophagy

p53 and JNK are two apoptosis-regulatory factors frequently deregulated in cancer cells and also involved in the modulation of autophagy. We have recently investigated the links between these two signalling pathways in terms of the regulation of autophagy. We showed that 2-methoxyestradiol (2-ME), an antitumoral compound, enhances autophagy and apoptosis in Ewing sarcoma cells through the activation of both p53 and JNK pathways. In this context, p53 regulates, at least partially, JNK activation which in turn modulates autophagy through two distinct mechanisms: on the one hand it promotes Bcl-2 phosphorylation resulting in the dissociation of the Beclin 1-Bcl-2 complex and on the other hand it leads to the upregulation of DRAM (Damage-Regulated Autophagy Modulator), a p53 target gene. The critical role of DRAM in 2-ME–mediated autophagy and apoptosis is underlined by the fact that its silencing efficiently prevents the induction of both processes. These findings not only report the interplay between JNK and p53 in the regulation of autophagy but also uncover the role of JNK activation in the regulation of DRAM, a pro-autophagic and pro-apoptotic protein.

Autophagy and microtubules – new story, old players

Summary Both at a basal level and after induction (especially in response to nutrient starvation), the function of autophagy is to allow cells to degrade and recycle damaged organelles, proteins and other biological constituents. Here, we focus on the role microtubules have in autophagosome formation, autophagosome transport across the cytoplasm and in the formation of autolysosomes. Recent insights into the exact relationship between autophagy and microtubules now point to the importance of microtubule dynamics, tubulin post-translational modifications and microtubule motors in the autophagy process. Such factors regulate signaling pathways that converge to stimulate autophagosome formation. They also orchestrate the movements of pre-autophagosomal structures and autophagosomes or more globally organize and localize immature and mature autophagosomes and lysosomes. Most of the factors that now appear to link microtubules to autophagosome formation or to autophagosome dynamics and fate were identified initially without the notion that sequestration, recruitment and/or interaction with microtubules contribute to their function. Spatial and temporal coordination of many stages in the life of autophagosomes thus underlines the integrative role of microtubules and progressively reveals hidden parts of the autophagy machinery.

Regulation of autophagy by amino acids and MTOR-dependent signal transduction

Amino acids not only participate in intermediary metabolism but also stimulate insulin-mechanistic target of rapamycin (MTOR)-mediated signal transduction which controls the major metabolic pathways. Among these is the pathway of autophagy which takes care of the degradation of long-lived proteins and of the elimination of damaged or functionally redundant organelles. Proper functioning of this process is essential for cell survival. Dysregulation of autophagy has been implicated in the etiology of several pathologies. The history of the studies on the interrelationship between amino acids, MTOR signaling and autophagy is the subject of this review. The mechanisms responsible for the stimulation of MTOR-mediated signaling, and the inhibition of autophagy, by amino acids have been studied intensively in the past but are still not completely clarified. Recent developments in this field are discussed.

c-Jun NH2-Terminal Kinase Activation Is Essential for DRAM-Dependent Induction of Autophagy and Apoptosis in 2-Methoxyestradiol–Treated Ewing Sarcoma Cells

Ewing sarcoma and osteosarcoma are two aggressive cancers that affect bones and soft tissues in children and adolescents. Despite multimodal therapy, patients with metastatic sarcoma have a poor prognosis, emphasizing a need for more effective treatment. We have shown previously that 2-methoxyestradiol (2-ME), an antitumoral compound, induces apoptosis in Ewing sarcoma cells through c-Jun NH(2)-terminal kinase (JNK) activation. In the present study, we provide evidence that 2-ME elicits macroautophagy, a process that participates in apoptotic responses, in a JNK-dependent manner, in Ewing sarcoma and osteosarcoma cells. We also found that the enhanced activation of JNK by 2-ME is partially regulated by p53, highlighting the relationship of JNK and autophagy to p53 signaling pathway. Furthermore, we showed that 2-ME up-regulates damage-regulated autophagy modulator (DRAM), a p53 target gene, in Ewing sarcoma cells through a mechanism that involves JNK activation. The silencing of DRAM expression reduced both apoptosis and autophagy triggered by 2-ME in Ewing sarcoma and osteosarcoma cells. Our results therefore identify JNK as a novel mediator of DRAM regulation. These findings suggest that 2-ME or other anticancer therapies that increase DRAM expression or function could be used to effectively treat sarcoma patients.

Glutamate dehydrogenase contributes to leucine sensing in the regulation of autophagy.

Amino acids, leucine in particular, are known to inhibit autophagy, at least in part by their ability to stimulate MTOR-mediated signaling. Evidence is presented showing that glutamate dehydrogenase, the central enzyme in amino acid catabolism, contributes to leucine sensing in the regulation of autophagy. The data suggest a dual mechanism by which glutamate dehydrogenase activity modulates autophagy, i.e., by activating MTORC1 and by limiting the formation of reactive oxygen species.

Reactive Oxygen Species, AMP-activated Protein Kinase, and the Transcription Cofactor p300 Regulate α-Tubulin Acetyltransferase-1 (αTAT-1/MEC-17)-dependent Microtubule Hyperacetylation during Cell Stress

Background: Tubulin acetylation is a hallmark of microtubule stabilization, which may modulate the binding of microtubule-associated proteins. Results: Microtubules are hyperacetylated because of stress-induced cellular signaling upstream of the tubulin acetyltransferase MEC-17/αTAT1. Conclusion: MEC-17/αTAT1 is regulated by p300, reactive oxygen species, and AMP-activated protein kinase. Significance: Microtubule hyperacetylation is important for cell adaptation to stress through autophagy induction and for cell survival. Beyond its presence in stable microtubules, tubulin acetylation can be boosted after UV exposure or after nutrient deprivation, but the mechanisms of microtubule hyperacetylation are still unknown. In this study, we show that this hyperacetylation is a common response to several cellular stresses that involves the stimulation of the major tubulin acetyltransferase MEC-17. We also demonstrate that the acetyltransferase p300 negatively regulates MEC-17 expression and is sequestered on microtubules upon stress. We further show that reactive oxygen species of mitochondrial origin are required for microtubule hyperacetylation by activating the AMP kinase, which in turn mediates MEC-17 phosphorylation upon stress. Finally, we show that preventing microtubule hyperacetylation by knocking down MEC-17 affects cell survival under stress conditions and starvation-induced autophagy, thereby pointing out the importance of this rapid modification as a broad cell response to stress.

Mutations in Two Zinc-Cluster Proteins Activate Alternative Respiratory and Gluconeogenic Pathways and Restore Senescence in Long-Lived Respiratory Mutants of Podospora anserina

In Podospora anserina, inactivation of the respiratory chain results in a spectacular life-span extension. This inactivation is accompanied by the induction of the alternative oxidase. Although the functional value of this response is evident, the mechanism behind it is far from understood. By screening suppressors able to reduce the life-span extension of cytochrome-deficient mutants, we identified mutations in two zinc-cluster proteins, RSE2 and RSE3, which are conserved in other ascomycetes. These mutations led to the overexpression of the genes encoding the alternative oxidase and the gluconeogenic enzymes, fructose-1, 6 biphosphatase, and pyruvate carboxykinase. Both RSE2 and RSE3 are required for the expression of these genes. We also show that, even in the absence of a respiratory deficiency, the wild-type RSE2 and RSE3 transcription factors are involved in life-span control and their inactivation retards aging. These data are discussed with respect to aging, the regulation of the alternative oxidase, and carbon metabolism.

Proteolysis of Pseudomonas exotoxin A within hepatic endosomes by cathepsins B and D produces fragments displaying in vitro ADP‐ribosylating and apoptotic effects

To assess Pseudomonas exotoxin A (ETA) compartmentalization, processing and cytotoxicity in vivo, we have studied the fate of internalized ETA with the use of the in vivo rodent liver model following toxin administration, cell‐free hepatic endosomes, and pure in vitro protease assays. ETA taken up into rat liver in vivo was rapidly associated with plasma membranes (5–30 min), internalized within endosomes (15–60 min), and later translocated into the cytosolic compartment (30–90 min). Coincident with endocytosis of intact ETA, in vivo association of the catalytic ETA‐A subunit and low molecular mass ETA‐A fragments was observed in the endosomal apparatus. After an in vitro proteolytic assay with an endosomal lysate and pure proteases, the ETA‐degrading activity was attributed to the luminal species of endosomal acidic cathepsins B and D, with the major cleavages generated in vitro occurring mainly within domain III of ETA‐A. Cell‐free endosomes preloaded in vivo with ETA intraluminally processed and extraluminally released intact ETA and ETA‐A in vitro in a pH‐dependent and ATP‐dependent manner. Rat hepatic cells underwent in vivo intrinsic apoptosis at a late stage of ETA infection, as assessed by the mitochondrial release of cytochrome c, caspase‐9 and caspase‐3 activation, and DNA fragmentation. In an in vitro assay, intact ETA induced ADP‐ribosylation of EF‐2 and mitochondrial release of cytochrome c, with the former effect being efficiently increased by a cathepsin B/cathepsin D pretreatment. The data show a novel processing pathway for internalized ETA, involving cathepsins B and D, resulting in the production of ETA fragments that may participate in cytotoxicity and mitochondrial dysfunction.

[Autophagy: a new concept in cancer research].

Macroautophagy is a lysosomal catabolic process involved in recycling cell components and maintaining cellular homeostasis. Identifying some of the molecules involved in the control and execution steps of autophagy has shed light on the close link between autophagy and tumour progression. Several tumour-suppressor proteins -including Beclin 1, a protein involved in autophagosome formation- positively regulate autophagy. Conversely, some oncogenic proteins display inhibitory effects on autophagy. The antitumoral role of autophagy is supported by its involvement in reducing chromosome instability, proliferation and inflammation of tumour cells. However, autophagy can also be a protumoral mechanism which helps tumour cells to adapt to changes in their microenvironment (hypoxia, starvation...). Moreover, autophagy is induced in response to several anticancer treatments. This response can either be a mechanism allowing cell survival or a mechanism promoting cell death. The aim of this article is to summarize recent progress focusing on the dual role of autophagy in cancer.