Introduction

From the previous chapters, we have learned that the neuroblastomas show heterogeneous clinical and biological features. Patients with localized tumor usually respond very well to the therapy, and infants show a better response to the treatment than older pediatric patients . Conversely, at least 50% of patients fall in the High-Risk (HR) group that usually respond poorly to the treatments, and show an overall survival lower than 40% after 5 years since diagnosis . Nowadays, the neuroblastoma study groups, including European Countries (SIOPEN – Société Internationale d’Oncologie Pédiatrique European Neuroblastoma, and GPOH – German Society for Pediatric Hematology/Oncology) and USA (COG – Children’s Oncology Group), have similar approaches to the treatment of HR patients, which are mainly based on: polychemotherapy, immunotherapy, radiotherapy, and autologous cell transplantation . More recently, SIOPEN, GPOH, and COG groups have proposed some new agents for targeted therapy. For instance, the COG have already used the Crizotinib , an Anaplastic Lymphoma Kinase (ALK) receptor tyrosine kinase (RTK) inhibitor, while SIOPEN and COG, in the near future, will probably introduce the LKB378, another ALK inhibitor proposed by Novartis. Even so, the rush for more effective and less toxic drugs against different molecular targets, including ALK receptor, continues , and will certainly bring additional changes in currently adopted therapeutic policies.

It is broadly known that chemotherapy and radiation therapy block the proliferation neuroblastoma cells. Moreover, chemotherapy is also able to induce apoptosis, leading to the elimination of tumor cells. More recently, another phenomenon that can evenly impact a course of neuroblastoma cell death, known as autophagy, has been described . Autophagy is a biological process that can be induced by a drug(s), and it can be found along with apoptosis activation. Nonetheless, the autophagy in cancer is also an effective mechanism of drug resistance, which may impede the complete drug activity, and be an obstacle for cancer cure . This aspect is getting particularly evident with the introduction of personalized therapy and the use of specific targeting agents. Recently, Aveic et al. have shown that autophagy is a defensive property of neuroblastoma cells, which can reduce the activity of ALK inhibitors Entrectinib and Crizotinib. In the following, several reports confirmed that we have to be aware of possible activation of the autophagic process when treating neuroblastoma . However, the current indications about autophagy in neuroblastoma are still elusive. Indeed, the role of autophagy in neuroblastoma cells should be more carefully evaluated in new trials, whether compounds inhibiting autophagy could be used as adjuvant therapy.

The Autophagy-Lysosome System

The term autophagy (from the Greek words “auto”—self, and “phagy”—to eat) was coined for the first time by Christian de Duve in 1963 during his seminar on the discovery of lysosomes, which brought him the Nobel Prize in Physiology and Medicine in 1974 . The autophagic phenomenon was described in the late 1950s, and until the 1990s the studies in this field were largely based on the morphological observations related to the formation of vesicles that were able to deliver intracellular components to the lysosomes . However, the molecular mechanisms underlying this catabolic process remained unknown until 1993, when Yoshinori Ohsumi described a genetic screen, in yeast, that brought the isolation of the first autophagy-related genes (ATG). ( Box 6.1 ).

In the last 2 decades, autophagy has been largely investigated from a morphological, biochemical, and molecular point of view. Nowadays, it is recognized as the major degradative system that eukaryotic cells use to digest portions of their cytosol . Three major forms of autophagy have been identified: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA), which are different in terms of physiological functions and the mode of cargo delivery to lysosomes . The microautophagy involves the direct uptake of small cytosolic elements through the invagination of the lysosomal membrane . The CMA requires the presence of the chaperones protein HSC70 (heat shock cognate 70), which brings the specific substrates, containing a KFERQ-like pentapeptide, into proximity of the lysosomal membrane and promote their internal translocation mediated by LAMP2 (lysosome-associated membrane protein 2) . The macroautophagy, simply referred as autophagy, denotes the general degradation of intracellular components through the fusion of the double membrane structures named autophagosomes with the lysosomes. It is the major regulatory catabolic mechanism in the cell, responsible for preserving cellular homeostasis. Autophagy occurs at a basal level in all cell types and efficiently ensures the regular turn-over of cytosolic components. It warrants a degradation of protein aggregates and dysfunctional organelles, which will be harmful to the cell. Moreover, autophagy has a prosurvival role. Under stress conditions, like nutrient deprivation and hypoxia, cells activate autophagy in order to degrade cytosolic components and generate free amino and fatty acids to supply the energy need .

The initial steps of autophagy involve the formation (nucleation) and expansion (elongation) of an isolation membrane, which is also named phagophore. The edges of the phagophore then fuse to form the autophagosome, a double-membrane vesicle that sequesters portion of cytosolic material; the autophagosome subsequentially fuses with a lysosome to form an autolysosome where the engulfed material, together with the inner membrane, is degraded ( Fig. 6.1 ).

Schematic representation of autophagy process.

A simplified version of three main autophagy steps: initiation —phagophore formation is trigged; elongation —double-membrane vesicle is closed, and autophagosome is created; and termination —the fusion of lysosome with autophagosome occurs generating autolysosome. The final result is degradation of cellular constituents (cargo), which then can be reused by the cell itself.

The major source of autophagosome membranes is the endoplasmic reticulum even if all membranous structures in the cell, like the plasma membrane, mitochondria and Golgi, could provide lipids to form the autophagosomes . Independently from their origin, all autophagy membranes contain the lipidated forms of the ubiquitin modifiers LC3s and GABARAPs .

Even if autophagy was initially described as a bulk degradative process, in the last decade it became clear that the autophagy machinery can target specific protein aggregates, organelles or pathogens, and selectively degrade them through the lysosomal system. Several targets of selective autophagy have been characterized: protein aggregates (aggrephagy), mitochondria (mitophagy), peroxisomes (pexophagy), ribosome (ribophagy), endoplasmic reticulum (ER-phagy), and pathogens (xenophagy) . The selectivity is given by the presence of the autophagy receptors. These proteins have the property to simultaneously bind the cargos and the autophagy modifiers (LC3s and GABARAPs) through their LC3-interacting regions. In mammalian cells, more than two dozen autophagy receptors have been identified. Each of them has its role in the selectivity of the cargo, and their common feature is to be degraded in the lysosomes together with their specific targets .

Molecular Mechanisms of Autophagy

Autophagy is highly inducible through a plethora of stress stimuli, especially during nutrient deprivation. The major signaling pathway involved during starvation is the mechanistic target of rapamycin (mTOR) signaling . mTOR is one of the key regulators of the autophagy process because it shuts down the entire system in the presence of growth factors and abundant amino acids . The mTOR complex 1 (mTORC1) phosphorylates and inactivates ULK1 and ATG13 in a standard nutrient situation. Upon autophagy induction, the ULK1 complex, which includes ULK1-ATG13-FIP200-ATG101, is activated and translocated to the endoplasmic reticulum where it promotes autophagosome membranes formation. ULK1 complex inhibition can be relieved upon mTORC1 inactivation by AMP-activated protein kinase (AMPK), which is sensitive to ATP consumption, and furthermore catalyzes the activating phosphorylation of ULK1. Once in the endoplasmic reticulum, the ULK1 complex regulates the class III phosphatidylinositol (PtdIns) 3-kinase complex (including Beclin 1, ATG14L, VPS15, VPS34, and AMBRA1). The phosphatidylinositol-3 phosphate (PtdIns3P) recruits double FYVE-containing protein 1 (DFCP1), and promotes the formation of specific endoplasmic reticulum structures, the omegasomes, where the autophagosomes generate. The subsequent expansion of the autophagosomes mainly relies on PtdIns3P production and PtdIns3P binding proteins of the WD-repeat domain phosphoinositide-interacting (WIPI) family. Finally, mature autophagosomes fuse with lysosomes forming the autolysosomes, where the engulfed cargo is degraded as a consequence of the luminal acidification and activation of lysosomal hydrolases . The recruitment of Beclin 1 to the PtdIns3-kinase complex is also sensitive to starvation. In nutrient-rich conditions, Beclin 1 is associated with Bcl-2, and under starvation, it is released upon Bcl-2 phosphorylation by JNK1 .

In addition to the mTORC1-ULK1-AMPK complexes, two ubiquitin-like conjugation systems and over 35 Atg genes in yeast, and more than a 100 ATG genes in mammals are responsible for autophagosome formation and maturation. One ubiquitin-like conjugation system involves the ubiquitin ligase ATG7, and it is responsible for the formation of a supramolecular protein complex containing ATG5, ATG12, and ATG16L. This complex has an important role in the elongation and closure of the isolated membrane. The second system, which includes ATG3, ATG4, and ATG7, promotes the lipidation of the LC3s and GABARAPs proteins. The ATG8 family members are cleaved and subsequently conjugated to a phosphatidylethanolamine (PE). Lipidated LC3s (LC3 II), as well as the other ubiquitin modifiers GABARAPs, are recruited to form autophagosomes, serve as adaptors for cargos to be degraded and have largely been investigated for monitoring autophagy flux in vitro and in vivo ( Fig. 6.2 ).

Autophagy-regulatory network.

The interaction network between the main autophagy regulators is generated using STRING: Functional protein association networks database. Several types of interconnection are included as indicated on the right side of the Figure. Small nodes indicate the proteins of unknown 3D structure, whereas large nodes indicate that some 3D structure is known or predicted for the protein.

Autophagy in Neuroblastoma

The involvement of autophagy in the regulation of tumorigenic processes is controversial. Since the time of de Duve in 1960s, many years passed before the impaired regulation of autophagy was related to carcinogenesis . This link came to light around the year 2000 when researchers realized that the autophagy role widens beyond the expected regulation of intracellular recycling of protein aggregates and dysfunctional organelles. In fact, being part of basic cellular mechanisms, autophagy is required for the maintenance of physiological conditions within the cells, and it is not surprising that its deregulation influences the general cell homeostasis . However, it is now clear that the intersection between autophagy regulation and tumor development is a complex and still not sufficiently understood topic. One of the main paradoxes, which still remain under debate, is whether autophagy sustains tumor formation or acts as a tumor suppressor mechanism . The most plausible hypothesis is that autophagy contribution to the tumorigenic process depends on the tumor stage. Autophagy could express tumor-suppressor properties in the early phases of tumor development, but it could also sustain tumor expansion at the advanced stages . In this last case, we are frequently dealing with the existence of disseminated and/or metastatic tumor cells that, by autophagy activation, can adapt to the conditions of the constant toxic insults due to the administered therapy.

As far as neuroblastoma is considered, a relatively small number of reports discussed the importance of autophagy in determining the characteristics of this tumor by the end of the year 2010. However, the interests in understanding the relationship between autophagy and neuroblastoma formation, or rather the connection between autophagy and impaired response to applied treatments, are bursting. Recently, for the first time, it has been reported through evidence that autophagy protein-regulators, in particular, LC3B and Beclin 1, are differentially expressed among the diverse stages of neuroblastoma tumors, or among primary tumors and metastatic masses . In particular, in the stage 4 tumors, a stronger staining signal for LC3B-II protein level was found more frequently, whereas the primary tumor masses diagnosed in patients above 1 year of age expressed significantly more Beclin 1 protein. It is therefore plausible that a better response to therapy observed in patients with stage 1 and 2 versus stage 4 neuroblastomas might be determined by the intensity of the cytoprotective autophagy in stage 4 tumors.

Interestingly, stage 4S tumors, which generally guarantee good prognosis to the patients, show low levels of autophagy proteins and have a high incidence of apoptotic events. It is an additional finding that suggests a relevance of autophagy in the tumorigenesis of neuroblasts.