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  • Review
  • Open Access

17 March 2023

Metastasis in Neuroblastoma and Its Link to Autophagy

1
School of Science and Technology, Nottingham Trent University, Clifton Site, Nottingham NG11 8NS, UK
2
Division of Cellular and Molecular Pathology, Department of Pathology, Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 0QQ, UK
Life2023, 13(3), 818;https://doi.org/10.3390/life13030818
This article belongs to the Special Issue The Role of Autophagy in Cancer
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Abstract

Neuroblastoma is a paediatric malignancy originating from the neural crest that commonly occurs in the abdomen and adrenal gland, leading to cancer-related deaths in children. Distant metastasis can be encountered at diagnosis in greater than half of these neuroblastoma patients. Autophagy, a self-degradative process, plays a key role in stress-related responses and the survival of cells and has been studied in neuroblastoma. Accordingly, in the early stages of metastasis, autophagy may suppress cancer cell invasion and migration, while its role may be reversed in later stages, and it may facilitate metastasis by enhancing cancer cell survival. To that end, a body of literature has revealed the mechanistic link between autophagy and metastasis in neuroblastoma in multiple steps of the metastatic cascade, including cancer cell invasion and migration, anoikis resistance, cancer cell dormancy, micrometastasis, and metastatic outbreak. This review aims to take a step forward and discuss the significance of multiple molecular players and compounds that may link autophagy to metastasis and map their function to various metastatic steps in neuroblastoma.

1. Introduction

Neuroblastoma (NB) accounts for close to 10% of all cancer cases in children but disproportionately leads to 15% of cancer-related deaths in this population of patients, equating to 1 in every 7000 live births [1]. The median age of diagnosis is usually 18 months, but it can be diagnosed in infants < 18 months, children 18 months–12 years, or adolescents older than 12 years of age [2,3]. NB is also regarded as one of the most common paediatric solid tumours relating to extracranial tissues and is thought to originate from the neural crest [3,4,5]. Furthermore, the site of presentation of this cancer varies from the abdomen, chest, neck, and pelvis, while over half of the patients will show metastasis to sites including the lymph nodes, bone, and bone marrow at the time of diagnosis [2,6,7,8].
Stages in NB can be categorised based on two systems: the international neuroblastoma risk group system (INRGSS) linked to image-defined risk factors (IDRF) and pre-treatment/surgery disease extent and comprise L1, L2, M and MS. On the other hand, the international neuroblastoma staging system (INSS) is linked to post-surgical disease extent and includes stages 1–4 and 4S (defined specifically in [3,9,10,11]). Briefly, the INRGSS L1 and L2 are both locoregional tumours, and L1 does not have IDRF, while L2 bears IDRF. Accordingly, IDRFs are defined as imaging surrogates of the tumour with regard to anatomical positions that predict a tumour resection success. M in this categorisation represents a widely spread disease, and MS (metastatic special) represents an age-specific (<18 months) localised disease (L1 and L2) linked to metastasis in locations including the skin, liver, and bone marrow (but not cortical bone) [3,9,10,11]. Further, the INSS stages 1 and 2 represent locoregional tumours having been completely or partially resected, respectively, and stage 3 denotes locoregional tumours of greater dimension which cross the midline and are deemed unresectable. Stages M and MS, however, represent distant metastasis and metastatic special, respectively. The latter denotes an age-specific (<12 months) category with a primary tumour of stage 1 and limited metastasis to the skin, liver and minimal bone marrow (but not cortical bone) [3,9,10,11].
Furthermore, patients can be stratified into low, intermediate, and high-risk, a classification based on various factors, including chromosomal alterations, MYCN status, stage, ploidy, age, and differentiation status, in addition to other criteria [3,12,13,14]. Concerning high-risk NB, MYCN amplification cases constitute approximately 40–50% of this group, and this genetic alteration is the strongest predictor of poor prognosis [3,12,13,14]. Additionally, high-risk NB may encompass two other subgroups, including alternating length of telomeres (ALT) (approximately 25%) and 23–31% of cases displaying telomere reverse transcriptase (TERT) gene rearrangements (e.g., rearrangement of 5p15.33, leading to strong enhancer insertion into TERT coding sequences) [9,15,16].
Risk groups also determine treatment stratification, ranging from surveillance to surgery for cases with low-risk stratification, response-adjusted doses of chemotherapy and surgery for intermediate-risk groups, and chemotherapy, surgery, radiotherapy, and myeloablative therapy for high-risk cases [13,14,17].
Tumour cell migration and invasion are key requirements for cancer metastasis and progression. Specifically, in the case of NB, metastasis to the lymph nodes, bone, and bone marrow accounts for most of the NB-related cancer progression and mortality [3,8,18]. To that effect, the clinical outcomes of NB patients with metastasis remain poor in contrast to patients with localised disease presentation that bear a more favourable overall survival (OS) projection [19]. OS is defined as the time post-diagnosis/treatment in which the patient is still alive. One good example of the comparison of prognoses is NB patients showing bone marrow metastasis compared to NB patients with no bone marrow metastasis, in which the former group displayed an OS of 35.87% compared to 87.7% in the latter cohort, respectively [20].
From a molecular outlook, tumour cells must acclimate to harsh and stressful conditions to metastasise, enter the systemic circulation, disseminate, and eventually colonise a distant site [21]. During these processes, tumour cells will face cellular stressors, including the depletion of nutrition and oxygen resources and cell–cell or cell–basement membrane contact loss, and these factors may be inducers of autophagy, a tightly regulated self-preservation mechanism facilitated by lysosomal autolysis [22,23]. Given this background, it stands to reason that autophagy may be closely linked to the process of metastasis, and accordingly, autophagy plays a dual role in suppressing or promoting cancer progression due to the removal of damaged proteins and cellular components or conferring resistance to stress, respectively [23,24]. From a metastasis standpoint, the role of autophagy may be examined closer in 4 arbitrary steps comprising the in-situ primary tumour, intravasation, extravasation and seeding in new tissue, and finally, colonisation of distant sites [8,21,23,25,26] (Figure 1).
Figure 1. The roles of autophagy in metastasis. (A) The primary tumour cells (for example, neuroblastoma cells) during the in-situ step may be affected by autophagy; for example, autophagy may inhibit metastasis by limiting pro-metastasis inflammatory responses and limiting growth (−), or it can promote metastasis by increasing drug resistance (+). (B) Tumour cells (for example, neuroblastoma cells) can enter the circulation, constituting circulating tumour cells, and autophagy promotes resistance to anoikis (anoikis is triggered by a contact loss with the basement membrane), hence promoting metastasis (+). Arrowhead showing tumour cells entering the circulation. (C) At the extravasation and seeding step, the circulating tumour cells enter a distant site, and autophagy may trigger the onset of dormancy in the upper panel of (C) (the cell with a slightly darker yellow colour represents a dormant neuroblastoma cell) or survival in the new microenvironment (+) and drug resistance (+) in the bottom panel of (C). Arrowhead showing tumour cells exiting the circulation. (D) In the distant metastasis site, autophagy can limit the expansion of dormant tumour cells (+/−) in the upper panel of (D) or promote adaptation to this new environment, expansion and metastatic outbreak (+) in the bottom panel of (D). It is noteworthy that (+) and (−) signs in the text signify a positive or negative contribution of autophagy to metastasis, respectively, while the grey basement layer in (A,B) and pink basement layer in (C,D) represent distinct tumour microenvironments before and after migration, respectively.
During step 1 (Figure 1A), autophagy may suppress metastasis by limiting tumour growth by reducing pro-metastasis immune cell infiltration. Equally, it can promote metastasis by promoting resistance to drug treatment, including TNF-related apoptosis-inducing ligand (TRAIL). In step 2 (Figure 1B), the tumour cells enter the circulation and thereby lose contact with the basement membrane, and the triggering of autophagy can suppress anoikis (cell death due to loss of cell–basement membrane contact). In step 3 (Figure 1C), surviving tumour cells (circulating tumour cells) may exit the circulation and seed within the distant tissue. Triggering autophagy may lead to resistance to drug treatment, including TRAIL, the survival of the tumour cells in the new microenvironment, or the onset of dormancy. Finally, in step 4 (Figure 1D), in the distant tissue, autophagy may limit the expansion of the dormant tumour cells or equally lead to a metastatic outbreak and expansion (reviewed in [8,21,23,25,26]). Given this background, this study aimed to take a step forward from describing mechanisms that link autophagy to metastasis and focus on examining the literature for molecular players and compounds that may link these processes, specifically in NB. In addition, for each molecular player and compound, the evidence supporting (a) its significance in NB, (b) the regulation of autophagy by this molecular player or compound in other cancers, and (c) whether it may be a suitable biomarker for predicting NB progression or may be used for NB treatment, will also be showcased and dissected.

2. Autophagy in NB Metastasis

2.1. The Molecular Basis of Autophagy

Autophagic processes involve a myriad of over 30 genes termed autophagy-related genes (ATGs) that contribute to the main steps of autophagy, including the initiation, the process of the phagophore nucleation, the elongation/maturation of a phagophore and autophagosome formation, and finally, the fusion of the lysosomes with the autophagosome [27,28]. To that end, phagophore initiation is orchestrated by the release of ULK1 from the ULK1 complex (ULK1/ATG13/FIP200/ATG101), and this process is dependent on the mTOR and AMPK signalling pathways (the induction step). For example, in low-glucose states, the ULK1 function may be altered by receiving an activating phosphorylation modification from AMP-activated kinase (AMPK). In contrast, in a high-glucose state, ULK1 receives inhibitory phosphorylation due to the activity of mTOR and raptor [29]. ULK1, post-activating phosphorylation, will activate the nucleation of the phagophore by phosphorylating Beclin 1. The recruitment of ATG9A and its associated proteins to the phagophore complex (nucleation) also ensues. These events lead to the formation of a phagophore (isolation membrane) coated by a complex of proteins inclusive of VPS15, VPS34, ATG14L and Beclin 1 (also known as the PI3K complex). Autophagosome elongation and maturation are mediated through the activation of ATG12 and cleavage of microtubule-associated protein light chain 3 (LC3) conjugation systems. As a result of these processes, the autophagosome is then coated with LC3-II-bound P62, and since P62 is degraded by the autolysosomes, hence the reduction of P62 is an indicator of autophagy progression. Finally, LC3-II levels are indicative of autophagosome formation since LC3-I is converted to LC3-II through lipidation processes [21,30,31] (Figure 2).
Figure 2. Multiple molecular steps of autophagy. A pre-autophagosomal structure is formed in the initiation stage and comprises ULK1, FIP200, ATG101 and ATG13, and in low glucose states, the ULK1 may receive activating phosphorylation via AMPK activity (whereas in the high-glucose state, ULK1 receives inhibitory phosphorylation due to mTOR and raptor activity, although this is not shown in the figure). ULK1 then activates the nucleation of the phagophore and the phosphorylation of Beclin 1. In the nucleation phase, ATG9A is recruited to the isolation membrane (phagophore), and the recruitment of ATG2 and ATG18 ensues. In addition, the isolation membrane is coated with VPS34, VPS15, ATG14L, and Beclin 1. Further, in the elongation and maturation phase, the processing of LC3 and ATG12 conjugation systems leads to the elongation of the phagophore and the formation of autophagosomes. It is noteworthy that the ATG12 conjugation system comprises ATG12, ATG7, ATG16L and ATG5 proteins. These processes contribute to the coating of the autophagosome with proteins such as LC3-II and P62, while the formed autophagosome may fuse with a lysosome in later stages.

2.2. Molecular Players and Compounds Linking Autophagy to Metastasis

The following sections will investigate 11 molecular players or compounds linking autophagy to metastasis in NB in several categories, including autophagy players, non-coding RNA and compounds. This information has been summarised in Table 1.
Table 1. The list of the 11 molecules and compounds linking autophagy to metastasis in NB.

2.2.1. Mediators of Autophagy and Their Link to NB Metastasis

UNC-51-like kinase1 (ULK1) is a serine/threonine kinase and forms a complex with ATG13, ATG101 and FIP200. AMPK can mark ULK1 with activating phosphorylation under energy and nutrient depletion conditions, and this will activate the nucleation of the phagophore by phosphorylating Beclin 1 and thereby advance the process of autophagy [43]. Therefore, ULK1 is a kinase that regulates the early steps of autophagy. Given this background, Dower and colleagues exposed a panel of NB cell lines, including SH-SY5Y and SK-N-AS cell lines, to a small molecule inhibitor of ULK1, SBI-0206965 [32]. This compound reduced autophagic flux in SK-N-AS cells under starvation conditions, decreased ULK1 and increased P62 protein levels (a protein accumulated when autophagy is inhibited) [44]. Autophagic flux is defined as the molecular process of autophagy, including autophagosome formation followed by the lysis of macromolecules [23]. In addition, this treatment led to a decreased LC3-II accumulation in the presence of bafilomycin A1 (a molecule that inhibits autophagosome-lysosome fusion) [32]. These proof-of-principle experiments revealed that SBI-0206965 reduced autophagic flux and LC3 lipidation. In addition, under both normal and starvation conditions, SBI-0206965 treatment led to the upregulation of cleaved poly-ADP ribose polymerase (PARP) and caspase-3 and consequently apoptosis (and annexin-V staining), while cell viability was decreased [32]. The effect on cytotoxicity was more profound under starvation conditions, presumably due to the dependence of NB cells on autophagy for survival under these conditions, hence collectively suggesting that the small molecule inhibitor suppressed autophagy but promoted apoptosis [32].
Further, the authors used a genetic model, SK-N-AS cells expressing a dominant-negative ULK1 gene (i.e., dnULK1K46N allele forming dnULK1-SK-N-AS cells), to show similar effects to SBI-0206965 on apoptosis. In evidence, dnULK1 increased P62, cleaved caspase-3 and PARP. In addition, the activity of caspase-3/7/8 increased, as did the percentage of annexin-V-expressing cells [32].
Consistently, the xenografting of SK-N-AS cells expressing stable dnULK1 or empty vectors to the NOD/SCID-gamma (NSG) mouse model was coupled with monitoring tumour growth using a luciferase reporter. This approach showed that dnULK1-SK-N-AS xenograft grew much slower, displayed P62 accumulation, and increased PARP and caspase-3 levels compared to empty vector controls [32]. Further, using a metastatic burden mouse model, SK-N-AS cells expressing stable dnULK1 or empty vector were xenografted to the bloodstream of mice via their tail vein; the former group survived for a significantly longer period and also displayed reduced liver metastasis burden, while the empty vector group displayed enlarged and fluid-filled livers [32].
Regarding anoikis, which is defined as a form of apoptosis due to prolonged detachment of cells from their basement membrane and extracellular matrix, both dnULK1 and SBI-0206965 treatments in SK-N-AS cells lead to increased caspase-3/7/8 activation, annexin-V and cleaved PARP, suggesting that the inhibition and targeting of ULK1 could perhaps lead to increased anoikis and result in metastasis suppression [32]. In addition, potential synergism between TRAIL and SBI-0206965 was investigated in SK-N-AS cells, and this combination led to increased apoptosis and TRAIL-sensitised SK-N-AS cells to SBI-0206965. TRAIL treatment, per se, increased autophagic flux, suggesting that perhaps autophagy has been upregulated to alleviate TRAIL-mediated apoptosis levels [32]. This study highlighted the therapeutic potential of ULK1 in NB and how autophagic flux may be linked to the metastasis of tumour cells (Figure 3).
Figure 3. The contribution of ULK1 to NB apoptosis and metastasis. (A) ULK1 forms complexes with ATG13, ATG101 and FIP200. ULK1 can be activated by AMPK via activating phosphorylation when glucose levels are low. ULK1 then can contribute to the activation of nucleation of the phagophore and the phosphorylation of Beclin 1. (B) The addition of SBI-0206965, a small molecule inhibitor of ULK1, led to the inhibition of autophagy marked by an increase in P62, a decrease in ULK1, LC3 lipidation, and LC3-II levels in the presence of bafilomycin A1. Therefore, SBI-0206965 decreased autophagic flux. (C) SBI-0206965 treatment in NB cell lines led to the upregulation of PARP and caspase-3, while cell viability was decreased, suggesting that the inhibition of ULK1 could lead to anoikis (hence ULK1 leads to anoikis resistance and perhaps metastasis). (D) The genetic inhibition model, SK-N-AS cells expressing a dominant-negative ULK1 gene (e.g., dnULK1K46N), similar to SBI-0206965, led to increased cleaved PARP, annexin-V, caspase-3 and enhanced enzymatic activity of caspase-3/7 and 8 and may also promote anoikis (hence the presence of ULK1 can lead to anoikis resistance). (E) Xenografting of SK-N-AS cells expressing stable dnULK1 led to P62 accumulation, increased PARP and caspase-3 levels, and a reduced metastasis burden in the liver in this group compared to their control counterparts. (F) TRAIL and SBI-0206965 combination in SK-N-AS cells led to increased apoptosis. TRAIL treatment, per se, increased autophagic flux, suggesting that perhaps autophagy has been upregulated to counter TRAIL-mediated apoptosis levels.

2.2.2. Non-Coding RNA with Oncogenic Roles Linked to Autophagy in NB Metastasis

Many studies have investigated the role of long non-coding RNAs (lncRNAs) in the NB pathogenesis and progression [45,46,47]. As evidence, in a study, the role of NORAD, a lncRNA, in the pathogenesis of NB was investigated, and the effect of this lncRNA on the proliferation, apoptosis, autophagy and metastasis was assessed [33]. Accordingly, NORAD was upregulated in NB tissues compared to matched healthy tissues, while the expression of NORAD was associated with advanced INSS stage and metastasis. In addition, the knockdown of NORAD using siRNA led to proliferation reduction in NB cell lines, including SK-N-SH, and IMR-32 [33]. In addition, NORAD knockdown led to the promotion of apoptosis and both aspects were validated on a protein level whereby NORAD knockdown reduced PCNA, cyclin D1 and Bcl-2 protein, while Bax protein was upregulated (PCNA and cyclin D testing proliferation, Bax and Bcl-2 testing apoptosis, and Bcl-2 is an apoptosis inhibitor). Invasion and transwell migration assays were also utilised to show that the knockdown of NORAD led to reduced migration and invasion capacity of the NB cells [33]. Furthermore, pertinent to the link between NORAD and autophagy, the authors observed that the knockdown of NORAD also increased the protein levels of Beclin 1, LC3-II/I ratio and ATG5 and a reduction of P62, suggesting that autophagic cell death was triggered as a result of NORAD depletion. Having established the baseline effect of knocking down NORAD, the authors attempted to establish the effect of NORAD knockdown on the chemoresistance of NB cell lines, inclusive of SK-N-SH and IMR-32 treated with Doxorubicin (DOX) [33]. This revealed a statistically significant reduction in NB proliferation, metastasis, and DOX resistance (evident by a decrease in the half-maximal inhibitory concentration (IC50) for NORAD-depleted samples). NORAD, therefore, effectively promoted resistance to DOX, metastasis and proliferation, whereas it inhibited autophagy and apoptosis [33].
Furthermore, using Starbase software, the authors revealed that miR-144-3p was a target of NORAD, and this was further verified using reporter assays, while it was also revealed that the overexpression of NORAD led to miR-144-3p downregulation [33]. This link prompted the authors to determine the mechanistic basis of NORAD-based cancer progression and DOX resistance through the potential sponging effect of miR-144-3p activity. Initially, the authors revealed that the depletion of NORAD led to enhanced miR-144-3p levels [33]. Further, the depletion of miR-144-3p (using anti-miR-144-3p) reduced the suppressive effects of siRNA-mediated NORAD depletion on NB cell proliferation, migration, metastasis, and DOX resistance; hence the NORAD exerted its oncogenic effects through the negative regulation of miR-144-3p. Furthermore, histone deacetylase 8 (HDAC8) was shown to be a target of miR-144-3p in NB since the protein levels of HDAC8 were increased or decreased as a result of anti-miR-144-3p treatment or miR-144-3p accumulation, respectively. Furthermore, NORAD depletion led to reduced HDAC8 expression while anti-miR-144-3p treatment recovered the expression of HDAC8; hence HDAC8 was regulated through the NORAD/miR-144-3p axis [33].
Finally, the authors also tested the effect of NORAD depletion on a murine xenograft model, showing that this treatment led to reduced tumour growth. Measuring protein levels in these in vivo samples also showed the reduction in NORAD and HDAC8 and the increase in miR-144-3p levels in NORAD-depleted samples. Overall, NORAD promoted proliferation, metastasis, and drug resistance, while it inhibited autophagy and apoptosis [33] (Figure 4).
Figure 4. NORAD-mediated NB progression. (A) The knockdown of lncRNA NORAD using siRNAs reduces proliferation, invasion, and DOX resistance in NB cell lines, including SK-N-SH and IMR-32. In addition, NORAD knockdown may lead to P62 downregulation and increased levels of Beclin 1, ATG5, LC3-II/LC3-I (LC3-II/1), and enhanced apoptosis. (B) miR-144-3p was a target of NORAD, depicted by the binding of NORAD (blue) to miR-144-3p (red). (C) Further, the overexpression of NORAD led to the downregulation of miR-144-3p, resulting in cancer progression and DOX resistance. (D) The depletion of miR-144-3p (red) using anti-miR-144-3p (purple) reduced the suppressive effects of siRNA-mediated NORAD depletion on NB cell proliferation, migration, metastasis, and DOX resistance (NORAD and NORAD siRNA have been depicted in blue and black colours, respectively). (E) Histone deacetylase 8 (HDAC8) was shown to be a target of miR-144-3p in NB. miR-144-3p expression (accumulation) led to the downregulation of protein levels of HDAC8, while anti-miR-144-3p treatment led to HDAC8 upregulation. In addition, NORAD depletion led to reduced HDAC8 expression, collectively suggesting HDAC8 was regulated through the NORAD/miR-144-3p axis. (F) NORAD depletion in a murine xenograft model led to reduced tumour growth.
Other oncogenes have also been studied in NB, including the role of hsa_circ_0013401, a circular RNA (circRNA), in proliferation, apoptosis, autophagy, and migration in this cancer. CircRNAs lack a cap structure at the 5′ ends and can therefore form circular structures. As evidence, hsa_circ_0013401 knockdown in NB cell lines, including SH-SY5Y and SK-N-BE, led to reduced proliferation and migration while apoptosis and autophagy were promoted [34]. The effect of hsa_circ_0013401 knockdown on apoptosis and autophagy was evident since the authors revealed an increase in the protein levels of Bax, cleaved caspase-3, LC3B-II/I ratio and Beclin 1. Accordingly, miR-195 was a target of hsa_circ_0013401, while PAK2 was a target of miR-195 (tested through reporter assays) and in a mouse xenograft model, hsa_circ_0013401 enhanced tumour formation and progression through the regulatory axis of miR-195/PAK2 [34]. In addition, the role of another lncRNA, SNHG16, in NB metastasis was investigated. This lncRNA was linked to poor clinical outcomes in NB patients. Furthermore, SNHG16 knockdown led to reduced proliferation and migration in NB cell lines (for example, SK-N-SH and IMR-32) and animal models [35]. This lncRNA formed a regulatory loop with ATG5 and miR-542-3p through which its effects on proliferation, migration and autophagy were implemented. Mechanistically, SNHG16 upregulated ATG5 and induced a sponging effect on miR-542-3p [35].

2.2.3. Non-Coding RNA with Tumour Suppressor Roles That Affect Autophagy in NB Metastasis

In another study, the role of MEG3 a nucleus-based lncRNA, in NB was investigated since this lncRNA was negatively linked with the NB INSS stage [36]. In addition, MEG3 was positively linked to NB patient survival and negatively linked to the unfavourable clinical characteristics of these patients. For example, NB patients with higher MEG3 expression displayed a greater 5-year event-free survival (EFS) along with overall survival, while the lower expression of MEG3 was linked to NB disease progression [36]. EFS refers to the period after treatment in which the patient has not suffered from any events which were intended to be prevented by the treatment. Further, the effect of the upregulation of MEG3 in SK-N-BE2, SK-N-AS and SH-SY5Y cell lines was assessed by assays including colony formation, EDU and CCK-8 and these experiments showed that MEG3 suppressed NB cell proliferation and reduced colony formation capacity [36]. Using transwell and wound-healing assays, MEG3 was shown to suppress cell migration and invasion [36]. Gene ontology analyses revealed that MEG3 was involved in metabolic and catalytic activities in addition to autophagy and mTOR signalling pathways. The link between MEG3 and autophagy was also tested by upregulating MEG3, resulting in reduced Beclin 1, ATG3, ATG12 and LC3-II/LC3-I ratios. In addition, the link between autophagy and mTOR was expected since mTOR is a known autophagy regulator; however, what was not fully understood was whether MEG3 regulated autophagy in an mTOR-linked fashion in NB, and to address this, two pieces of evidence were generated [36]. Firstly, the overexpression of MEG3 inactivated mTORC1, implying that MEG3 may have affected autophagy in an mTOR-independent fashion. This statement was fortified by observing that rapamycin (an mTOR pathway inhibitor) did not affect NB autophagy (but affected migration). Secondly, the FOXO1 inhibitor, AS1842856, suppressed autophagy (e.g., lowered LC3), while the overexpression of MEG3 suppressed FOXO1 expression. This evidence collectively led to the conclusion that MEG3 regulated autophagy through FOXO1 modulation but not through the mTOR signalling pathway [36].
Finally, the downregulation of MEG3 (by using a MEG3 silencer) led to NB malignant characteristics, including enhanced proliferation, migration, and invasion established using transwell assays in an mTOR-dependent fashion, while the silencing of MEG3 increased autophagy markers, including ATG16, Beclin 1 and ATG3, collectively bringing into light the role of MEG3 as a negative regulator of autophagy and metastasis in NB [36] (Figure 5).
Figure 5. The role of MEG3 in regulating autophagy in NB metastasis. (A) MEG3, a nucleus-based lncRNA, was tested in NB cell lines, including SK-N-BE2, SK-N-AS, and SH-SY5Y. The overexpression of MEG3 led to reduced cell proliferation, colony formation, migration, and invasion capacity. In addition, MEG3 overexpression reduced autophagy protein levels, including ATG3, ATG12, and Beclin 1, but this effect was not implemented through mTORC1 since in MEG3-overexpressing NB cells, mTORC1 was inactivated. (B) A FOXO1 inhibitor (AS1842856) mimicked MEG3 overexpression and led to reduced LC3 protein levels (suppression of autophagy), while the overexpression of MEG3 suppressed FOXO1, suggesting perhaps that MEG3 attenuated autophagy through FOXO1 regulation. MEG3 downregulation led to enhanced levels of ATG16, ATG3, and Beclin 1 (in an mTOR-independent manner), while low levels of MEG3 enhanced migration through mTOR signalling.
Other tumour suppressor non-coding RNAs, including miR-34a, have also been investigated in the context of NB metastasis [37]. This non-coding RNA was suppressed in NB cell lines (for example, SH-SY5Y and SK-N-SH) and patient tissue linked with reduced viability and survival, respectively. miR-34a targeted ATG5 and thereby suppressed proliferation, apoptosis, metastasis and autophagy. This was evident since restoring ATG5 rescued the inhibitory effect of miR-34a on these processes [37].

2.2.4. Compounds and Small Molecule Inhibitors That Link Autophagy in NB Metastasis

In another study, the effect of Isatin (a derivative of indirubin with anti-tumour properties) on SH-SY5Y NB cells was investigated using microarray analyses revealing 429 differentially expressed genes. Further, gene ontology analysis revealed that these differentially expressed genes displayed roles in redox activities, transcription, transport and metabolism [38]. Further, KEGG analysis of the differentially expressed gene list revealed the involvement of Isatin-regulated genes in chemokine and mTOR signalling and ribosome-related pathways [38]. A subset of genes relating to mTOR signalling, including DDIT4, RHEB, EIF4EBP1 and RPS6KB1, were taken forward for further analysis, and the differential expression of these genes was verified using RT-qPCR [38]. The link between the mTOR pathways and NB metastasis and invasion was also investigated in SH-SY5Y cells. Isatin reduced the invasion capacity of SH-SY5Y, tested through migration and invasion assays [38]. From a mechanistic viewpoint, Isatin reduced metastasis through the inhibition of mTOR phosphorylation while it increased the phosphorylation levels of AMPK, the inhibitor kinase of mTOR. Since autophagy is linked to mTOR signalling, the authors also tested the expression levels of LC3-II, Beclin1, and P62. Isatin treatment led to LC3-II and Beclin1 upregulation, and P62 downregulation, suggesting the induction of autophagy [38]. In effect, Isatin suppressed NB metastasis while triggering higher levels of autophagy markers and mTOR dysregulation. This suggested that autophagy is closely linked to metastasis in NB. Overall, it could be argued that Isatin exerted inhibitory effects on NB metastasis through autophagy-related and mTOR signalling pathways [38] (Figure 6).
Figure 6. Isatin reduced the invasion and metastasis capacity of NB cells. (A) SH-SY5Y cells were treated with Isatin and were subjected to microarray analysis, revealing the differential expression of genes involved in redox activities, transcription, transport, metabolism, chemokine and mTOR signalling, and ribosome-related pathways. (B) Isatin treatment led to the reduced invasion and migration capacity of SH-SY5Y NB cells through the inhibition of phosphorylated-mTOR (p-mTOR) and the increase of phosphorylated-AMPK (p-AMPK) (since AMPK inhibits mTOR). (C) Isatin treatment led to the upregulation of LC3-II and Beclin1 and the downregulation of P62.
The role of apatinib, a vascular endothelial growth factor receptor-2 (VEGFR-2) small molecule inhibitor in inducing cell cycle arrest and apoptosis in a panel of NB cells, including BE(2)-M17, IMR-32 and SH-SY5Y, was assessed [39]. As evidence, this study used a CCK-8 and colony-forming assays to test for viability and proliferation across different doses of apatinib, and this compound was shown to reduce NB cell viability, proliferation, and colony formation. In addition, apatinib reduced the expression of Ki-67, a proliferation marker, in these cells. Further, apatinib treatment led to the arrest of these cells in the G0/G1 phase and the increase in apoptotic cells in both BE(2)-M17 and SH-SY5Y cell lines; this was evident since the levels of both Bcl-2/Bax ratio (an apoptosis direction indicator), and cyclin D1 levels were reduced [39]. Thus, apatinib induced apoptosis and cell cycle arrest while inhibiting cell viability and colony formation. Furthermore, using both transwell and wound healing assays in BE(2)-M17 and SH-SY5Y cell lines, apatinib significantly reduced migration potential in these cells. Mechanistically, bioinformatic analyses showed that apatinib could affect the PI3K/AKT and MAPK/ERK pathways, while this was also established in vitro since this molecule downregulated p-AKT, p-mTOR and p-ERK protein levels [39]. Finally, the LC3B-II/I ratio (LC3B-II is an indicator of the number of autophagosomes, while LC3B is the main isoform of LC3) and ATG5 protein levels were increased as a result of apatinib treatment of SH-SY5Y and BE(2)-M17 NB cells [39,48].
It could be argued this study revealed that apatinib treatment could induce apoptosis and autophagy while suppressing metastasis through the PI3K/AKT/mTOR and MAPK/ERK signalling axis [39]. However, the direct link between the suppression of VEGFR2 by apatinib and the in vivo reduction of metastasis was not investigated, but since apatinib inhibits p-mTOR, this may be the logical explanation for this compound’s capacity to reduce NB migration [39] (Figure 7). In agreement with these studies, Honokiol was also shown to induce apoptosis and autophagy (for example, it increased LC3-II levels in neuro-2a NB cell line) and reduce migration, and these effects were linked to the PI3K/Akt/mTOR pathway [40].
Figure 7. Apatinib reduced the invasion and metastasis capacity of NB cells. (A) NB cell lines inclusive of BE(2)-M17, IMR-32 and SH-SY5Y were treated with apatinib leading to reduced viability, colony formation, migration and proliferation of these cells while increasing apoptosis rate (reduced Bcl-2/Bax ratio). (B) Apatinib treatment led to cell cycle arrest (reduced cyclin D1 levels) while decreasing phosphorylated mTOR, AKT, and ERK (p-mTOR, p-AKT and p-ERK, respectively). (C) LC3-II/I and ATG5 protein levels were increased as a result of apatinib treatment, revealing autophagy activation.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Data sharing is not applicable.

Conflicts of Interest

The author declares no conflict of interest.

Abbreviations

ALTAlternating length of telomeres
AMPKAMP-activated kinase
ATGsAutophagy-related genes
DOXDoxorubicin
EFSEvent-free survival
EMTMesenchymal transition
HCQHydroxychloroquine (HCQ)
HDAC8Histone deacetylase 8
IC50Half-maximal inhibitory concentration
IDRFImage-defined risk factors
INRGSSInternational neuroblastoma risk group system
INSSInternational neuroblastoma staging system
LC3microtubule-associated protein light chain 3
LncRNAsLong non-coding RNAs
LRPPRCMitochondrion-associated autophagy inhibitor
3-MA3-Methyladenine
NBNeuroblastoma
NSGNOD/SCID-gamma
OSOverall survival
PARPPoly-ADP ribose polymerase
TERTTelomere reverse transcriptase
TRAILTNF-related apoptosis-inducing ligand
VEGFR-2Vascular endothelial growth factor receptor 2
ULK1UNC-51-like kinase1

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