Neuroblastoma (NB) is the most common extracranial solid tumor in children. It most often occurs in the adrenal gland, accounting for approximately 15% of all pediatric cancer-related deaths (1, 2). Although active multimodal treatment is adopted, the prognosis of children in different stages of disease still varies greatly (3). At present, it is generally believed that children with distant metastasis often have a poor prognosis. Nevertheless, there is a special stage–stage MS–that exhibits a phenomenon of spontaneous regression and a good prognosis (4).

To date, it is believed that the spontaneous regression of stage MS NB may involve the following mechanisms: (1) neurotrophin deficiency, (2) telomerase inactivation, (3) humoral or cellular immunity and (4) changes in epigenetic regulation(4). However, these mechanisms are still in the preliminary research stage, and the spontaneous regression of stage MS NB has not been fully explained.

The modes of cell death include accidental cell death and regulatory cell death (RCD). At present, many evidences show that RCD is the main factor of cell death, which is a spontaneous mode of cell death, including apoptosis, autophagy, ferroptosis and other modes, and is closely related to tumor progression (5, 6). Here, we speculate that the spontaneous regression of stage MS NB may be related to RCD.

Apoptosis is the most deeply studied RCD mode at present. The process of apoptosis eventually activates caspase protein, resulting in cell death (11). The weakening of apoptosis often leads to tumorigenesis, and the overexpression of antiapoptotic oncogenes such as BCL-2/BCL-XL, MCL1 or the IAP proteins is conducive to the survival of tumor cells (12). The results of our study showed that BIRC5, SLCO4A1, POPDC3, and HK2 were involved in the process of apoptosis.

BIRC5, also known as survivin, is the strongest inhibitor of apoptosis found thus far (13), and its main role is related to the inhibition of caspase activity (14). The expression of BIRC5 is downregulated during normal tissue development and cannot be detected in most final differentiated adult tissues (15). In a variety of tumors, such as ovarian cancer, breast cancer, colorectal cancer (16) and renal cell carcinoma (17), the expression is increased and is positively correlated with metastasis and a low survival rate. SLCO4A1 is proved to be highly expressed in colorectal cancer and affect prognosis (18), but its role in NB has not been studied. POPDC3 belongs to the Popeye domain containing (POPDC) family, and is a recently discovered cyclic 3′,5′-adenosine monophosphate (cAMP) effector protein (19). Recent studies have shown that the expression level of POPDC3 is closely related to the cell proliferation, metastasis and prognosis of a variety of cancers, especially gastric cancer, and can be used as a potential cancer treatment target (20–22). In addition, research on POPDC3 covers topics including muscular dystrophy, cardiac function and other aspects (23, 24); research on POPDC3 in NB is lacking, but this is a topic worthy of in-depth exploration. HK2, hexokinase 2, is an important glycolytic enzyme that catalyzes the conversion of glucose to glucose 6-phosphate. Studies have shown that the expression level and activity of HK2 in metastatic NB tumor tissues are higher than those in local NB tumor tissues, suggesting that HK2 plays an important role in the formation of the malignant phenotype of NB and affects the progression of the disease (25).

This study showed that BIRC5, SLCO4A1, POPDC3, and HK2 were significantly downregulated in stage MS NB and affected the survival rate of children, indicating that the low expression of these four genes is a factor conducive to tumor regression. All these factors are involved in the process of apoptosis. We propose that the spontaneous regression of stage MS NB is closely related to the process of apoptosis, and the above four genes play an important role in this process of apoptosis.

In addition, our results show that BIRC5 and HK2 are also involved in autophagy. Autophagy is a cellular pathway involved in the degradation of proteins and organelles, and it is the mechanism of cell survival under stress stimulation. Currently, autophagy is considered to play a dual role in cancer. It inhibits the growth of benign tumors but promotes the growth of advanced tumors. Many research groups have established autophagy as a potential therapeutic target for cancer (26). A study showed that some autophagy-related genes were differentially expressed between stage MS and stage M (<18 months) NB. The results showed that autophagy inhibited the progression and promoted the spontaneous regression of NB (27).

It is generally believed that apoptosis and autophagy are not completely isolated processes (28). BIRC5 is an important molecule connecting the two processes (29). BIRC5 is positively regulated by the AKT/mTOR pathway to inhibit autophagy and apoptosis and promote tumor cell survival (14). The degradation of BIRC5 releases bound beclin-1, enhances autophagy and induces cell death, which also shows that the increase in BIRC5 levels is closely related to the inhibition of autophagy (30). In addition, some studies have pointed out that HK2 plays the same role as BIRC5 and participates in the process of autophagy and apoptosis through the AKT/mTOR pathway. Targeting HK2 to treat cancer is also a promising strategy (31–33). This is consistent with our results (BIRC5 and HK2 are downregulated genes), so we speculate that the spontaneous regression of stage MS NB is likely to be related to the autophagy and apoptosis processes related to BIRC5 and HK2 because when the expression levels of BIRC5 and HK2 are low, the inhibition of apoptosis and autophagy by other factors will be relieved.

TF, transferrin, was the only gene upregulated in stage MS NB in our results. It is a key molecule involved in ferroptosis. Its function is to transport iron from the site where heme is absorbed and degraded to the site where heme is stored and utilized. TF and its receptor TFR can regulate the process of ferroptosis directly or indirectly (34).

Ferroptosis is a newly defined RCD mode that was first proposed by Scott J. Dixon and others in 2012 (35). Ferroptosis is associated with a variety of pathological conditions, such as acute tissue injury, infection, inflammation, cancer and neurodegeneration (36, 37). The molecules involved in ferroptosis include Nrf2, p53, heme oxygenase‐1, FANCD2, and BECN1, and the role of ferroptosis in breast cancer, hepatocellular carcinoma, renal cancer and other diseases has been studied (38). Our study shows that the TF gene is differentially expressed between stage MS and stage M (<18 months) NB and affects the survival of children. We propose that the spontaneous regression of stage MS NB involves ferroptosis regulation and that TF plays an important role in this process.

GO enrichment and KEGG pathway analysis revealed that the DEGs were mainly involved in response to drug, response to xenobiotic stimulus and other processes, as well as in amebiasis, protein digestion and absorption, ECM-receptor interaction and other pathways. Among the above five DEGs that affect survival, both TF and HK2 are involved in the HIF-1 signaling pathway; TF is involved in the process of cell ferroptosis; and HK2 is involved in the processes of autophagy and apoptosis.

HIF-1 is a member of the hypoxia inducible factor protein family, which can help cells adapt to the hypoxic environment. HIF-1 is a signaling center, and its role in cancer has been fully described. Inhibiting HIF-1 is a promising treatment strategy for cancer and cancer-related inflammation (39, 40). The function of HIF-1 in NB has also been widely studied. The increased expression and activity of HIF-1 promotes the proliferation, invasion and migration of NB cells, which is positively correlated with the malignant degree of NB (41). Some studies have shown that HIF-1/HK2 can synergistically promote the development of breast cancer (42). However, this synergistic effect has not been studied in NB. In addition, in studies of different kinds of cells, it has been shown that an increase in HIF-1 levels can inhibit ferroptosis, and inactivation of this pathway can induce ferroptosis (43, 44). In osteoclasts, elevated HIF-1 expression can inhibit not only ferroptosis but also autophagy (45). Therefore, we think that the spontaneous regression of stage MS NB involves autophagy and ferroptosis regulated by TF, HK2 and the HIF-1 signaling pathway.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

The studies involving human participants were reviewed and approved by Children’s Hospital of Chongqing Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the minor(s)’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.

All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.