Autophagy and Novel Therapeutic Strategies in Neuroblastoma

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 ).

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 .