A Review of the Regulatory Mechanisms of N-Myc on Cell Cycle

Neuroblastoma has obvious heterogeneity. It is one of the few undifferentiated malignant tumors that can spontaneously degenerate into completely benign tumors. However, for its high-risk type, even with various intensive treatment options, the prognosis is still unsatisfactory. At the same time, a large number of research data show that the abnormal amplification and high-level expression of the MYCN gene are positively correlated with the malignant progression, poor prognosis, and mortality of neuroblastoma. In this context, this article explores the role of the N-Myc, MYCN gene expression product on its target genes related to the cell cycle and reveals its regulatory network in promoting tumor proliferation and malignant progression. We hope it can provide ideas and direction for the research and development of drugs targeting N-Myc and its downstream target genes.


Introduction
Neuroblastoma (NB) is the most common extracranial tumor in children. Nearly half of all NBs occur in infants under 2 years of age [1]. NB accounts for approximately 6-10% of childhood tumors and 5% of tumor mortality in 1-year-old children [1]. According to different high-risk factors, NB can be divided into the low-risk group, intermediaterisk group, and high-risk group [2]. For high-risk patients with NB, the prognosis is still unsatisfactory even if various intensive treatment options are combined [3,4]. It is worth noting that a large number of clinical data have shown that patients with high-risk neuroblastoma are often accompanied by abnormal amplification and high-level expression of the MYCN gene [5,6], suggesting that the MYCN gene plays an important role in tumor malignancy and development. Therefore, we took neuroblastoma as an entry point to summarize the potential mechanism of N-Myc regulation on the cell cycle, and also considered the regulation network in other tumor cells, in an attempt to complete the regulatory network of N-Myc on the cell cycle.
The MYCN gene is an important member of the MYC family. The MYC family is a group of oncogenes that was discovered earlier and has been studied extensively. It consists of three members: MYC, MYCN, and MYCL. All of them belong to the basic helix-loop-helix leucine zipper (bHLHLZ) DNA binding protein superfamily, and their products are nuclear DNA binding proteins [7,8], which usually act as transcriptional regulators and affect various processes such as the cell cycle, differentiation, angiogenesis, etc., by regulating a variety of genes [9]. Among them, the MYCN gene has certain temporal and spatial expression specificities. It is usually expressed in pre-B cells, kidneys, forebrain, hindbrain, and intestines during embryogenesis, and is highest in the developing brain [10]. N-Myc can regulate the expression of various target genes that are related to the cell cycle to affect the proliferation and development of tumor cells. The methods of regulation can be summarized as the following: First, the most classic regulation method is that N-Myc binds to the HLH-Zip of the chaperone MYC-associated protein X (MAX) in the nucleus to form a complex through the HLH-Zip structure, and acts on the E-box region that exists on the regulatory sequence of its target gene to regulate the expression of target genes [12]. N-Myc preferentially combines with E-box CATGTG and classic CACGTG. However, under the conditions of MYCN amplification, its specificity is reduced, and it can bind to other non-classical E-box motifs, including CTTTG, CACTTG, and CAACTG) [13]. It should be noted that although the E-box region on the gene is considered to be the classic scope of the MYC family, the E-box region where c-Myc can act does not mean that N-Myc can also act. In addition to MAX, N-Myc can also interact with WD repeat domain 5 (WDR5), which is a subunit of the histone H3K4 methyltransferase complex [14], H3K9me3/me2 demethylase, lysine demethylase 4B (KDM4B) [15], lysine demethylase 1A (KDM1A/LSD1) [16], etc., to regulate gene expression. Second, N-Myc can act independently on the E-box region of the transcription promoter of the target genes and directly regulate their expression [17]. Third, under the condition of gene co-expression, when both p53 and MYCN are expressed at high levels, N-Myc (MYC BOX II/IIIa region) can interact with the C-terminus of the tetramerized p53 protein (only tetrameric p53 can bind to chromatin) to form a complex, and this effect has nothing to do with the formation of N-Myc and MAX heterodimer mentioned above. The complex can regulate gene expression by acting on the E-box region or p53 response elements (p53-Res) [18]. Fourth, the antisense mRNA transcribed by MYCN itself can regulate its own expression by acting on the gene or its transcribed mRNA [19], indirectly affecting the expression of downstream genes. Therefore, N-Myc can affect the expression level of cell cycle-related target genes in a variety of ways, which is closely related to cell malignant proliferation.
Cyclins or the cyclin-dependent kinase (CDK) play a vital role in cell cycle regulation [20]. Cyclins are synthesized and degraded periodically as the cell cycle progresses, and they all have a conservative sequence (cyclin box) composed of approximately 100 N-Myc can regulate the expression of various target genes that are related to the cell cycle to affect the proliferation and development of tumor cells. The methods of regulation can be summarized as the following: First, the most classic regulation method is that N-Myc binds to the HLH-Zip of the chaperone MYC-associated protein X (MAX) in the nucleus to form a complex through the HLH-Zip structure, and acts on the E-box region that exists on the regulatory sequence of its target gene to regulate the expression of target genes [12]. N-Myc preferentially combines with E-box CATGTG and classic CACGTG. However, under the conditions of MYCN amplification, its specificity is reduced, and it can bind to other non-classical E-box motifs, including CTTTG, CACTTG, and CAACTG) [13]. It should be noted that although the E-box region on the gene is considered to be the classic scope of the MYC family, the E-box region where c-Myc can act does not mean that N-Myc can also act. In addition to MAX, N-Myc can also interact with WD repeat domain 5 (WDR5), which is a subunit of the histone H3K4 methyltransferase complex [14], H3K9me3/me2 demethylase, lysine demethylase 4B (KDM4B) [15], lysine demethylase 1A (KDM1A/LSD1) [16], etc., to regulate gene expression. Second, N-Myc can act independently on the E-box region of the transcription promoter of the target genes and directly regulate their expression [17]. Third, under the condition of gene co-expression, when both p53 and MYCN are expressed at high levels, N-Myc (MYC BOX II/IIIa region) can interact with the C-terminus of the tetramerized p53 protein (only tetrameric p53 can bind to chromatin) to form a complex, and this effect has nothing to do with the formation of N-Myc and MAX heterodimer mentioned above. The complex can regulate gene expression by acting on the E-box region or p53 response elements (p53-Res) [18]. Fourth, the antisense mRNA transcribed by MYCN itself can regulate its own expression by acting on the gene or its transcribed mRNA [19], indirectly affecting the expression of downstream genes. Therefore, N-Myc can affect the expression level of cell cycle-related target genes in a variety of ways, which is closely related to cell malignant proliferation.
Cyclins or the cyclin-dependent kinase (CDK) play a vital role in cell cycle regulation [20]. Cyclins are synthesized and degraded periodically as the cell cycle progresses, and they all have a conservative sequence (cyclin box) composed of approximately 100 amino acids, which mediates the combination of cyclin and CDK to form a complex and regulate the cell cycle [21]. CDK must be combined with cyclin to exhibit kinase activity. Therefore, the orderly appearance and degradation of cyclin can effectively regulate the activity of CDK, thereby initiating or regulating events such as DNA replication, mitosis, and cytokinesis [22]. The cyclin-dependent kinase inhibitor (CKI) can interact with cyclin and the CDK complex and inactivate it by changing the structure of the active site of CDK. CKI includes two families, the CDK-interacting protein/kinase inhibition protein (CIP/KIP) and inhibitors of CDK (INK), and is primarily involved in the regulation of G1 and S phases [23]. In addition, during the progression of the cell cycle, cyclin and CDK need to be accumulated, combined, activated to complete their normal physiological functions, and finally, degraded. The Skp1-Cullin 1-F Box (SCF) and the anaphase promotion complex (APC), both of which are ubiquitin ligase, link ubiquitin to G1/S and M phase cyclins and CKIs to degrade, thereby regulating the cell cycle [23].
In the beginning, we comprehensively referred to several papers on transcriptomic analysis of MYCN regulatory gene expression and screened a wide range of potential target genes [24]. However, transcriptomic analysis lacked strong evidence to prove the direct targeting of MYCN for these genes, so we further referred to articles related to experimental research to support the results.

The Target Genes of N-Myc
The target genes of N-Myc below are closely related to the cell cycle, and some of their products can act as transcription factors or combine with them to directly affect the expression of the key factors of the cell cycle, such as Cyclins and CDK, while some of them can regulate the activity and stability of cell-cycle-related proteins by exerting the activity of enzymes. In general, we screened 10 N-Myc target genes and listed them in the order of cell cycle progression (G1-M) according to their main mode of action. At the same time, the regulatory mechanisms of these target genes on the cell cycle are summarized (Supplementary Materials Table S1) and form a downstream signal regulatory network of N-Myc ( Figure 2).  The mechanism of N-Myc target genes regulating cell cycle progression. The ten target genes listed on the right side can be divided into three parts. First, the expression products of genes such as POU5F1, PTK2, PRMT1, MDM2, FOXM1, and DKC1 can act as or bind to transcription factors to regulate the expression of cell cycle-related genes (primarily including CDKs and Cyclins). Second, they serve as kinase to regulate the activity of proteins participating in cell cycle, such as PTK2, VRK1, and PLK1. Third, they play a vital role in balancing the stability of cell-cycle-related proteins, such as SKP2, MDM2, and PLAGL2.

POU5F1
POU class 5 homeobox 1 (POU5F1, also knowns as OCT4) is a homeodomain transcription factor of the Pit-Oct-Unc (POU) family, which is necessary for inducing the pluripotency of human and mouse somatic cells [25]. Pou5f1 can bind to the octamer motif binding site in the enhancer region or target promoter to activate the transcription and expression of target genes [26]. Bioinformatics analysis shows that POU5F1 is overexpressed in a variety of cancers [27].
Experiments have shown that endogenous N-Myc binds directly to the E-box site in the POU5F1 promoter to enhance its transcription. Furthermore, Pou5f1 binds to the first Figure 2. The mechanism of N-Myc target genes regulating cell cycle progression. The ten target genes listed on the right side can be divided into three parts. First, the expression products of genes such as POU5F1, PTK2, PRMT1, MDM2, FOXM1, and DKC1 can act as or bind to transcription factors to regulate the expression of cell cycle-related genes (primarily including CDKs and Cyclins). Second, they serve as kinase to regulate the activity of proteins participating in cell cycle, such as PTK2, VRK1, and PLK1. Third, they play a vital role in balancing the stability of cell-cycle-related proteins, such as SKP2, MDM2, and PLAGL2.

POU5F1
POU class 5 homeobox 1 (POU5F1, also knowns as OCT4) is a homeodomain transcription factor of the Pit-Oct-Unc (POU) family, which is necessary for inducing the pluripotency of human and mouse somatic cells [25]. Pou5f1 can bind to the octamer motif binding site in the enhancer region or target promoter to activate the transcription and expression of target genes [26]. Bioinformatics analysis shows that POU5F1 is overexpressed in a variety of cancers [27].
Experiments have shown that endogenous N-Myc binds directly to the E-box site in the POU5F1 promoter to enhance its transcription. Furthermore, Pou5f1 binds to the first intron of MYCN to promote MYCN transcription, so the two can form a positive feedback loop [28].
The overexpression of POU5F1 in BE(2)-C human NB type I cells can promote cell proliferation and increase its colony formation rate [29]. There are many mechanisms for Pou5f1 to regulate the cell cycle.
First, in adult stem cells or cancer cells, Pou5f1 up-regulates the expression of CCND by binding to the octamer motif in the CCND promoter region, thereby promoting the G1/S transition [30][31][32].
In addition, Pou5f1 can bind to the conservative promoter of miR-302 and up-regulate its expression [33]. miR-302 (over a certain threshold concentration) simultaneously suppressed the activities of CDK2 and cyclin D1/D2 to inactivate both complexes (cyclin D-CDK4/6 and cyclin E-CDK2), thereby blocking both G1-S transition pathways. miR-302 suppresses BMI-1 to slightly stimulate p16Ink4a/p14ARF expression, which binds to CDK4/6 and reduces its activity, thereby inhibiting the phosphorylation of retinoblastoma protein RB and eventually blocking a series of genes to prevent the entry of the S phase [34].
According to the classical model, in the early and middle G1 phase, Pou5f1 downregulates the protein phosphatase-1 (PP1) by promoting the expression of PP1 inhibitor nuclear inhibitor of protein phosphatase 1 (NIPP1) and CCNF, which inhibit the dephosphorylation of RB by PP1 [35]. Pou5f1 can also up-regulate CDK4/6-Cyclin D to promote the hypo-phosphorylation of RB. In mammalian cells, the physiological early 2 factor (E2F) exists as an E2F/dimerization partner (DP) heterodimer. E2F binds to DNA with DP proteins through an E2 recognition site and regulates gene transcription. RB can bind to E2F/DP to form a ternary complex to inhibit E2F activity [36]. When RB is hypo-phosphorylated, part of RB is released, restoring the transcriptional activity of E2F to up-regulate CCNE1/2 expression [37]. Cyclin E binds to and activates CDK2, which drives the hyperphosphorylation of RB and causes stronger effects, thereby driving the restricted point passage and increasing the transcription required for S phase entry. [37]. In addition, A new study suggests that CDK4/6-Cyclin D can only monophosphorylate the RB protein, leaving it still connected to E2F and inhibiting its activity. An unknown event can activate Cyclin E-CDK2 and further phosphorylate RB, thereby activating E2F. The mechanism of this new model remains to be further studied [37,38].
In addition, RB can directly bind to the central domain of the forkhead box M1 (FOXM1), thereby indirectly inhibiting the transactivation domain of FOXM1 and inhibiting the effect of FoxM1 as a transcription factor up-regulating the expression of POU5F1 [39].
In the G2/M phase, Pou5f1 can inhibit CDK1 activation by inhibiting the cell division cycle 25 (Cdc25) [38], resulting in a prolonged duration of the G2 phase, which is conducive to subsequent genome integrity checks and reducing chromosomal segregation [40].
Research shows that silencing POU5F1 significantly reduced the expression of the deacetylase the silent information regulator factor 2-related enzyme 1 (SIRT1), resulting in increased acetylation of p53 at K120 and K164, thereby maintaining the stability of p53 [38]. In addition, Pou5f1 can bind to the promoter region of integrin subunit α6 (CD49f), whose overexpression is sufficient to regulate cellular proliferation via regulation of the PI3K/AKT/p53 pathway [41]. p21 is a downstream target of p53, which can inhibit the activation of CDKs, leading to G1 and G2 blockade. Pou5f1 can also inhibit the activity of p21 by directly binding to the p21 promoter region or indirectly up-regulating DNA methyltransferase 1 (DNMT1), which is a major DNA methyltransferase responsible for maintaining methylation status during DNA replication [42].

PRMT1
Protein arginine N-methyltransferase 1 (PRMT1) is the most important asymmetric arginine methyltransferase in the human body. It plays a role in transcriptional co-activation by dimethylating histone H4 at R3 (H4R3me2as) [43]. PRMT1 is highly expressed in a variety of tumors and is positively correlated with tumor growth, progression, and poor prognosis [44][45][46].
N-Myc can up-regulate the expression of PRMT1 by acting on the region near the transcription promoter of the PRMT1 gene [47]. At the same time, the down-regulation of MYCN mRNA caused by the knockdown of PRMT1 suggests that PRMT1 may regulate the expression of MYCN at the transcriptional level [48]. In addition, studies have shown that PRMT1 enhanced the stability and expression of MYCN by methylating N-Myc protein at R65, which is related to CDK-mediated phosphorylation of N-Myc at S62 [48].
In breast cancer cells, PRMT1 can interact with CCAAT/enhancer-binding protein alpha (C/EBPα), a member of the leucine zipper transcription factor family, and methylate it at both R35 and R156 (the most critical sites) residues. The methylation of C/EBPα prevents its interaction with the inhibitor histone deacetylase 3 (HDAC3) and reduces the formation of the C/EBPα-HDAC3 complex, which has an inhibitory effect on Cyclin D1, thereby promoting the proliferation of tumor cells [45]. C/EBPα can act on the promoter region of the CCND1 gene to negatively regulate its expression. At the same time, C/EBPα can also exert a stronger inhibitory effect by recruiting HDAC3 to the promoter region of CCND1 [45].
In addition, studies have shown that PRMT1 can regulate the expression of a series of cell-cycle-related genes, including CCNA2, CCNB1, CCND1, CCNE2, CDK6, CDC20, and CDC23 [45]. The mechanism is still unclear, but it proved that PRMT1 was closely related to cell cycle regulation. However, in NB, PRMT1 promotes cell survival by up-regulating the expression of activating transcription factor 5 (ATF5) and inhibiting apoptosis, instead of regulating the cell cycle [49].

VRK1
Vaccinia-related kinase 1 (VRK1) is a member of the Ser/Thr kinase family. VRK1 can phosphorylate a variety of transcription factors including p53, and it can also cooperate with the c-Jun NH2-terminal kinase (JNK) pathway through the phosphorylation of c-Jun and activating transcription factor 2 (ATF2) to participate in cellular stress response [50]. VRK1 is highly expressed in a variety of tumor cells and is positively correlated with tumor cell proliferation, tumor progression, and poor prognosis [51][52][53][54].
VRK1 has been identified as the transcription target of MYCN, its gene transcription promoter region contains an E-box region, and its expression can be up-regulated by MYCN. At the same time, the analysis showed that in MYCN-amplified NB cells, the binding site of N-Myc presented a hypomethylated state (the degree of methylation is negatively correlated with the degree of gene expression), proving that VRK1 is regulated by MYCN. In addition, the down-regulation of VRK1 can also down-regulate the expression of MYCN, suggesting that there may be a positive feedback mechanism between the two [53].
High expression of VRK1 promotes the proliferation of NB cells [53]. Experiments showed that the proliferation of NB cells depends on the expression of VRK1. Observed via mRNA and immunohistochemistry, in NB and patient-derived tumor xenograft (PDX)derived cells, there is a strong positive correlation between the expression of VRK1 and that of the proliferation marker Ki67, as well as the mitotic index in the tumor. Concomitantly with this, a moderate knockdown of VRK1 will induce the down-regulation of cell-cycleprogressing protein levels (such as Cyclin D1 or MDM2) and lead to an increase in cell cycle inhibitors (such as p53 and its target p21) [53]. From the late G1 phase to the early S phase, Molecules 2023, 28, 1141 6 of 27 VRK1 phosphorylates the cAMP-response-element binding protein (CREB), which causes CREB to bind to the cAMP response element (CRE) of the CCND1 promoter and activate its transcription [55]. In liver cancer cells, the knockdown of VRK1 also up-regulates the expression of p27, which, in turn, causes cell G1/S phase arrest [56]. In addition, VRK1 is also necessary for exiting G0 and entering G1 [57].
In esophageal squamous cell carcinoma, the knockdown of VRK1 leads to the downregulation of the barrier-to-autointegration factor 1 (BANF1) [58]. BANF1 plays an important role in mitotic nuclear reorganization and directly affects cell proliferation. In addition, BANF1 can be used as a high-affinity substrate of VRK1 to be catalyzed and phosphorylated by VRK1, thereby weakening the interaction between BANF1 and DNA, destroying the connection between DNA and the nuclear membrane and maintaining the normal process of the cell cycle, resulting in a change in the process of cell mitosis [59,60]. Studies have shown that histone H2A T120 is phosphorylated by hVRK1 (human VRK1) in and around the CCND1 promoter. H2A T120 phosphorylation antagonizes H2A K119 ubiquitination and promotes H3 K4 methylation, thereby up-regulating CCND1 and promoting the oncogenic transformation of cells [61].
In head and neck squamous cell carcinoma, the VRK1 protein is positively correlated with several proliferation-related proteins including CDK2, CDK6, Cdc2, Cyclin B1/A, topoisomerase II (TOP2), survivin, and Ki67, suggesting that VRK1 can regulate the cell cycle [54]. VRK1 can act on the promoter regions of CDK2 and BIRC5 (also known as survivin) genes, up-regulate their expression, and regulate the cell cycle [54].

SKP2
The S-phase kinase-associated protein 2 (SKP2) gene is located in the 5pl3 region of the human chromosome, and the protein encoded by it is composed of 436 amino acid residues, also known as the p45 protein. SKP2 is sequentially linked by the F-box sequence, linker sequence, and protein-protein interaction module [62,63]. SKP2 is overexpressed in a variety of tumors and is positively correlated with tumor progression and poor prognosis [64][65][66].
Studies have shown that in MYCN-amplified NB cells, N-Myc can increase the activity of the SKP2 gene promoter and promote its expression by acting on the classic or nonclassical E-box region of the SKP2 gene transcription promoter region [67]. In MYCNamplified NB cells, N-Myc eliminates the repressive pRB-E2F transcription factor 1 (E2F1) complex bound to the SKP2 promoter by inducing CDK4 and up-regulates the expression of SKP2 [68].
The SKP2 gene contains a functional E2F response element (hSRE2), which is involved in the activation of the SKP2 promoter function. It is also required for the high-level expression of the SKP2 gene in many human tumor cell lines [69]. At the same time, the expression level of SKP2 is also regulated by RB. The combination of RB and E2F can inhibit the transcription of SKP2. In the G0 phase and the early stage of the G1 phase, RB can inhibit the expression of the Skp2 protein by keeping Skp2 and APC/CCdh1 in close proximity [70]. Both of these effects will keep Skp2 at a low level. In the late stage of the G1 phase, RB is phosphorylated and releases E2F and APC/CCdh1, causing SKP2 to be induced to be transcribed, and at the same time, its degradation efficiency is reduced, causing the cell to pass through the R point and enter the S phase [71].
A large number of studies have shown that there is an automatic induction circuit of Skp2. In this loop, the ectopic expression of SKP2 triggers the degradation of p27, leading to the activation of Cyclin E-CDK2, which was originally in an inhibited state by binding to p27. The activated CDK2 causes the phosphorylation of RB, and the release of E2F leads to the increased expression of SKP2 [71][72][73]. Mitogenic stimulation initiates the Skp2 automatic induction circuit by inducing Cyclin D1, activating CDK4/6, and inactivating RB [74]. Exogenous anti-mitogens, such as hyaluronic acid, may close the circuit by inhibiting the effects of Cyclin D1 and CDK4/6 [75].
A new mechanism for SKP2 to drive cell-cycle progression has been proposed. In the late stage of the G1 phase, activated Cyclin E-CDK2 phosphorylates Erα, triggering ERαSCF Skp2 binding and E2F1 transactivation. E2F1 further induces Cyclin E, Cyclin A, and Skp2, and drives the entry of the late G1 and S phases [76].
Skp2 is primarily related to the G1/S process. It targets cell cycle inhibitors (such as p27 Kip1 , p21 Cip1 , etc.) for ubiquitination and degradation, thereby promoting the cell cycle process [62,63,77]. It is a part of the SCF ubiquitin ligase E3 complex (SCF) and can link ubiquitin and induce the degradation of S phase kinase cyclin A and CDKI. Knockdown of SKP2 can significantly inhibit the growth and proliferation of MYCN-amplified or nonamplified NB cells. Studies have shown that the G1/S phase arrest of NB cells caused by the down-regulation of the Skp2 protein is positively correlated with the stability of the p27 protein. If the accumulation of p27 caused by the down-regulation of SKP2 is inhibited, the degree of cell cycle arrest will be reduced, indicating that the down-regulation of SKP2 causes the accumulation of p27, which causes tumor cells to arrest in the G1/S phase [63,67].

PTK2
Protein tyrosine kinase 2 (PTK2, also called adhesion focus kinase, FAK) is a nonreceptor cytoplasmic protein tyrosine kinase, which participates in the control of many signaling pathways by integrating signals from integrin and growth factor receptors, affecting cell proliferation, viability, movement, and survival. It has become the target of a variety of malignant tumors [78].
There are two N-Myc binding sites in the PTK2 promoter sequence. The electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) proved that N-Myc bound to the E-box in the PTK2 promoter [79]. Dual luciferase analysis showed that the activity of the PTK2 promoter was significantly increased in MYCN-amplified NB cells. A polymerase chain reaction (PCR) and Western Blot confirmed that mRNA and protein levels were elevated in NB cell lines along with elevated MYCN levels. In summary, MYCN can up-regulate PTK2 gene expression [79].
In MYCN-amplified NB cells, after inhibiting the expression of PTK2 with two PTK2 small-molecule inhibitors, the proliferation of tumor cells was significantly reduced, the percentage of cells in the G1 phase was significantly increased, and that in the S phase was decreased [80]. It showed that inhibiting PTK2 can inhibit the proliferation of NB cells and cause the cells to fail to pass the cell cycle and arrest in the G1 phase [80].
PTK2 is an important cell-signaling scaffold. When PTK2 is phosphorylated, it can activate downstream pathways, such as sarcoma family kinases (SFKs) and extracellular signal-regulated kinase (ERK), and then participate in the regulation of cell cycle processes [81]. In fibroblasts, PTK2 can enhance the activity of the transcription factor E26 transformation-specific B (EtsB) and the CCND1 promoter, up-regulating the expression of CCND1 [82]. Ets family transcription factors are specific downstream substrates of ERK. Experimental data showed that PTK2 regulated the transcription of CCND1 primarily through the activation of the ERK pathway [82]. As a target gene of PTK2, Krüppel-like transcription factor 8 (KLF8), a member of the family of transcription factors, was positively regulated by PTK2 [83]. The promoter sequence of CCND1 has been identified as the target of KLF8. CCND1 can be directly activated by the combination of KLF8 and GT box A, or by inhibiting the potential inhibitory regulator of Cyclin D1 [83]. In glioblastoma cells, high PTK2 expression can increase the expression of CCND1 and CCNE, reduce the expression of CDKN1B (p27 Kip1 ) and p21 Waf1 , and enhance the activity of CDK4, thereby promoting the G1/S phase transformation of glioblastoma cells [84].

DKC1
Dyskerin pseudouridine synthase 1 (DKC1) is a conservative X-linked gene encoding the RNA-binding protein Dyskerin (pseudouridine synthase). Dyskerin is an important part of telomerase. Dyskerin can bind to and stabilize small nucleolar RNAs-H/ACA box snoRNAs, thereby directing rRNA modification and playing a role in the processing of rRNA precursors [85]. In clear cell renal cell carcinoma (ccRCC) [86], glioma [87], NB [88], and liver cancer [89], DKC1 expression is up-regulated and promotes tumor progression, but it can also act as a tumor suppressor [90].
Experiments have shown that in the BE(2)-C cell line, N-Myc interacted with its chaperone MAX to form a complex, acting on a site near the DKC1 transcription promoter, which includes a non-canonical E-box region containing a CpG island downstream of the promoter, and two canonical E-box regions further downstream but still within the first intron region. It can up-regulate the expression of the DKC1 gene [88].
The ribosomal stress caused by the down-regulation of Dyskerin in NB with siRNA is the main reason for the stagnation of tumor cell proliferation [88]. In glioma cells, Dyskerin negatively regulates the expression of CDK2 and CCNE2, leading to G1 arrest [87]. In mouse thymocytes, the genetic interaction between DKC1 and p27 is required for limited cell cycle progression [91].
However, in pituitary tumors, impaired DKC1 function can affect the translation of specific mRNAs containing internal ribosome entry site (IRES) elements, including tumor suppressor p27. The p27 IRES element mediates the assembly of the 48S translation pre-initiation complex, thereby promoting the occurrence of pituitary tumors [90].

MDM2
The mouse double minute-2 (MDM2) gene belongs to the RING-finger protein family and is widely known as an oncogene. It is amplified in a variety of human cancers including NB [92,93] and is involved in a variety of key cell growth regulation processes [94].
In NB, N-Myc directly binds to the E-box in the MDM2 promoter and promotes the transcription of MDM2 [95]. In addition, when MDM2 transfers from the nucleus to the cytoplasm, it combines with the AU-rich elements in the MYCN 3 untranslated region (3 -UTR) to regulate the stability of MYCN mRNA and its translation, so the interaction between MYCN and MDM2 forms a positive feedback regulatory loop [96].
In normal cells, the overexpression of MDM2 can induce G1 phase arrest, but in many cancer cells, including cells overexpressing MDM2, the arrest in the G1 phase is not apparent. Mdm2 contains three growth-inhibitory domains, the overexpression of which can inhibit the proliferation of normal cells, but cancer cells overexpressing MDM2 can make the growth-inhibitory domains unable to perform their normal functions through a variety of ways, thereby evading the G1 phase block [97,98]. Experiments have shown that the Mdm2 protein mediated the ubiquitination and degradation of p53 and inhibited the function of p53 to activate the transcription of target genes, thereby reversing p53-mediated cell cycle arrest [99][100][101]. The growth suppressor p14/p19 interacts with Mdm2 to inhibit Mdm2-mediated ubiquitination and degradation of p53, thereby restoring the regulation of the cell cycle by p53 [102][103][104]. Mdm2 can also act as a bridge between RB and p53, forming an RB-MDM2-p53 trimer, thereby preventing the degradation of p53 [105].
In prostate cancer cells, Mdm2 competes with E3 ubiquitin ligase SCF Skp2 to bind to E2F1 and inhibits the ubiquitination of E2F1, thereby up-regulating the protein level of E2F1 [106]. E2F1 is considered a carcinogen due to its activity promoting cell cycle progression. In the early stage of G0/G1, RB binds to E2F1 to inhibit its transcriptional function [106,107]. When RB is phosphorylated, it can dissociate from E2F1, thereby activating the transcription of downstream target genes related to the cell cycle and enabling cells in the late G1 phase to initiate cell cycle progression [106,107].

FOXM1
FoxM1 belongs to the mammalian fox family transcription factor and has homology in its winged helix DNA binding domain [108]. FoxM1 plays a vital role in ensuring the fidelity of the cell division process. Inhibition of FoxM1 activity can lead to serious abnormalities during mitosis, such as frequent chromosome segregation, cytokinesis defects, and overt aneuploidy [109]. FOXM1 is up-regulated in a variety of cancers and plays a carcinogenic role in tumorigenesis [110].
Studies have shown that N-Myc can directly bind to the promoter of FOXM1 and up-regulate the expression of FOXM1 at the transcriptional level in NB cells [111].
FoxM1 is a pro-proliferation transcription factor that promotes cell cycle progression during the transitional phase of G1/S and G2/M [112]. In NB, knocking down FOXM1 can cause a significant increase in the proportion of cells in the G1 phase and a decrease in the S phase [113]. Many studies have shown that FoxM1 directly or indirectly regulated the activation of target genes, such as CCND, CDK4, and CCNE, that CDK2 induces cells to enter the S phase, Cyclin A-CDK1 is involved in the G2/M transition, and Cyclin B-CDK1 induces the entry of the M phase and promotes the process of mitosis [109,[114][115][116]. FoxM1 up-regulates CCNB1 and CCND1 expression by directly activating their corresponding promoters [117]. In addition, some key mitotic regulators, such as Cdc25B, polo-like kinase 1 (PLK1), aurora kinase B (AURKB), and centromere protein F (CENPF), are also regulated by FoxM1 [118]. Together, these indicate that FOXM1 regulates cell cycle progression primarily by regulating the expression of cell-cycle-related proteins.
FoxM1 and PLK have mutual regulatory effects. In the S/G2 phase, FoxM1 is phosphorylated by CDK1, which is the key to the interaction between PLK1 and FoxM1. In the G2/M phase, PLK1 combines with FoxM1 and directly phosphorylates it, thereby activating it to promote the expression of downstream mitotic regulators, including PLK1 itself [119]. This regulation forms a positive feedback loop, ensuring an orderly mitosis process [119].

PLK1
PLK1 is an important member of the serine/threonine protein kinase family, and its expression is elevated in a variety of human cancers [120]. It can antagonize apoptosis and increase the invasiveness of cancer cells and is positively correlated with poor prognosis [120]. In the DNA damage response, overexpressed PLK1 triggers the activation of CDK1 in a Cdc25A-dependent manner, and the activated CDK1 covers the checkpoint with damaged DNA during the phase transition, causing tumorigenesis [121].
In NB cells with MYCN amplified, N-Myc can directly activate PLK1 to transcribe and promote its expression [122]. PLK1 specifically binds to SCF Fbxw7 ubiquitin ligase to phosphorylate it and promotes its ubiquitination and proteasome degradation, thereby resisting the Fbxw7-mediated degradation of N-Myc, enhancing the stability of N-Myc protein, and constituting a positive feedback loop [122].
The expression of PLK1 is cell cycle dependent [123]. PLK1 can promote cell cycle progression by regulating multiple steps in the process of mitosis [124]. PLK1-dependent phosphorylation plays a critical role in mitotic spindle formation at the onset of mitosis [125]. PLK1 also controls mesoscopic maturation and is a necessary kinase for kinesin spindle protein (Eg5)-dependent separation of centrosome [126,127].
During the G2/M transition, Cdc25C is phosphorylated and activated by PLK1 and then activates CDK1 by dephosphorylating it, promoting the formation of the Cyclin B1-CDK1 complex and ensuring mitotic entry [128]. Secondly, PLK1 catalyzes the phosphorylation of Wee1 at S53, leading to the E3 ubiquitin ligase-dependent degradation of Wee1. Furthermore, PLK1 catalyzes the phosphorylation of myelin transcription factor 1 (Myt1) at S426, leading to the inhibition of its kinase activity. Both chemical modifications could promote the activation of CDK1 [128][129][130]. PLK1 further phosphorylates Cyclin B1 at S133 to promote the translocation of the Cyclin B1-CDK1 complex to the nucleus, thereby triggering the G2/M transition [131].
PLK1 plays a vital part in regulating sister chromatid separation. During the prophase, PLK1 phosphorylates the SA2 subunit of cohesins, resulting in the dissociation of a large number of cohesins from sister chromatids [132]. A small fraction of the cohesins remains on the sister chromatids, which is conducted by the interaction of protein phosphatase 2A (PP2A) with shugoshin 1 (Sgo1), to ensure chromosomal pairing until the end of metaphase [132][133][134]. Early mitotic inhibitor-1 (Emi1) binds to the Cdc20 subunit of APC/Cdc20 and prevents it from binding to its substrate separation enzyme [135]. PLK1 causes the phosphorylation of Emi1 and its degradation, thereby promoting metaphase and activating APC/C [135,136]. In addition, PLK1 directly phosphorylates and activates APC/C [137]. Activated APC/C causes the activation of the isolated enzyme, which in turn causes the sister chromatids to move toward the poles, leading to anaphase [137,138]. In addition, PLK1 is essential to error-free chromosome segregation by regulating the interaction between cytoplasmic linker protein 170 (CLIP-170) and microtubules [139].
PLK1 has an important effect on initiating cytokinesis. PLK1 directly binds and phosphorylates the centralspindlin subunit HsCYK4 at S157 during the formation of the intermediate zone, promoting the localization of the Rho-GTPase exchange factor (ECT2) in the intermediate zone to form a cleavage trench, marking the beginning of cytokinesis [140]. The initiation of cytokinesis is related to the formation of the polycomb repressive complex 1 (PRC1) complex [141]. CDK1 phosphorylates PRC1 and prevents PLK1 from binding to PRC1 [141]. In addition, PLK1 negatively regulates PRC1 by directly phosphorylating PRC1 at T602 to prevent premature cytokinesis [142].

PLAGL2
Polymorphic adenoma-like protein 2 (PLAGL2) is a zinc finger protein of the PLAG gene family. There are seven C2H2 zinc finger domains at the N-terminus, which are highly conserved and can bind to DNA and enable the transcription factor PLAGL2 to activate the transcription of specific genes [143]. PLAGL2 is related to a variety of malignant tumors, including lipoblastoma, hepatocellular carcinoma, glioma, colorectal cancer, and acute myeloid leukemia [144][145][146][147].
PLAGL2 promotes the transcription of MYCN by directly binding to specific sequences upstream of the coding sequence (CDS) of the MYCN gene, and N-Myc regulates the transcription of PLAGL2 through binding to five N-Myc E-boxes in the PLAGL2 promoter region, which forms a positive feedback regulation [148]. Experiments showed that the mRNA level of p53 family member TP73 was significantly increased in the PCR array screening of PLAGL2 expression, suggesting that PLAGL2 is involved in inducing the transcriptional activation of TP73 [149]. In cells expressing PLAGL2, it was observed that the expression levels of TP73 and its downstream target cell cycle inhibitors p21 and p57 increased [149]. In U937 cells, the expression of PLAGL2 inhibited cell proliferation and induced the G1 phase arrest and a small degree of arrest in the G2/M phase. When TP73 was knocked down, cells in the G1 phase decreased and cells in the S phase increased. This indicates that PLAGL2 may induce cell cycle arrest by regulating TP73 to affect cell cycle inhibitors [149].
In addition, other studies have shown that p53-induced RING-H2, Pirh2, formed a dimer through its N-terminus and C-terminus [150]. PLAGL2 can interact with Pirh2 dimers, which can inhibit the degradation of Pirh2 mediated by the proteasome. Pirh2 is the target gene of p53 and is up-regulated by p53, but the Pirh2 protein can negatively regulate the level and stability of p53 [150]. Experiments have shown that after silencing Pirh2, it will cause an increase in p53 levels, an increase in the portion of cells in the G1 phase, and a decrease in the S phase. Therefore, PLAGL2 may regulate p53 by interacting with Pirh2 and indirectly participate in the regulation of the cell cycle [150].

Another Seven N-Myc Target Genes
There are another seven N-Myc target genes according to the initial screening. These target genes have obvious effects on cell cycle regulation and tumor cell proliferation, which also have the potential to serve as targets for anti-tumor drugs. In order to facilitate reading and understanding, we summarized the regulations and mechanisms of these target genes in Table 1. However, the specific regulatory mechanism in the cell is currently unclear, and more in-depth research is needed in the future. Table 1. Target genes up-regulated by N-Myc and their regulating targets. The seven target genes listed in the form show their ability to regulate the process of cell cycle in a relatively unspecific way. Up-or down-regulation of the expression of these MYCN target genes affects the expression of genes further downstream, but there is no direct evidence of the regulatory mechanism.

Target Gene Phase Regulating Target
TERT [88] S [151] Inhibition of TERT leads to DDR and down-regulation of CCND1. The high expression of TERT significantly up-regulated the expression of CCND1 [151,152].
PBK [168] G2/M [169,170], M [171] PBK forms a complex with Cyclin B1-CDK1 and phosphorylates PRC1 in a Cyclin B1-CDK1-dependent manner, thereby participating in the formation of the mitotic spindle and promotes cytokinesis [171]. PBK interacts with p53 containing the DBD domain to down-regulate the transactivation function of TP53. Over-expression of PBK down-regulates the expression of p21 by reducing the recruitment of p21 promoter to p53 [169,170,172].

GLDC
Glycine decarboxylase (GLDC) is an environment-dependent metabolic oncogene. GLDC drives the occurrence of non-small cell lung cancer (NSCLC) and regulates the proliferation of cancer cells by promoting pyrimidine biosynthesis, glycolysis, and sarcosine production [174]. In gastric cancer, GLDC acts as a tumor suppressor gene, and the hypermethylation of its promoter makes it silent at the transcriptional level and promotes the occurrence of gastric cancer [175].
In MYCN-amplified NB cell lines, MYCN overexpression significantly increased the expression of GLDC [157]. Sequence detection and ChIP-qPCR revealed that N-Myc bound to the E-boxes in the GLDC promoter region and the first intron [157]. In summary, MYCN regulates the expression of GLDC in NB at the transcriptional level.
Knockdown of GLDC caused cell proliferation inhibition and G1 phase arrest [157]. Experiments have shown that down-regulation of the GLDC gene led to a significant reduction in the mRNA expression of cyclins and CDKs, including CCNA2, CCNB1, CDK1, CDK2, CCND1, CCNE1, DNA polymerase epsilon 2 (POLE2), and minichromosome maintenance complex component 5 (MCM5), which induced G1 blockade and inhibited cell proliferation [157]. It also caused changes in a variety of metabolic pathways, among which a significant reduction in purine and cholesterol synthesis can decrease the expression of cyclins and CDKs and inhibit cell proliferation [157].
GLDC is the P protein in the glycine cleavage system and can be combined with glycine to transfer the methylamine group of glycine to the T protein [158]. GLDC participates in the first and rate-limiting step of glycine decomposition [157]. It catalyzes the conversion of glycine into carbon dioxide, ammonia, and 5,10-methylenetetrahydrofolate (CH2-THF) [159]. However, CH2-THF drives the new synthesis of thymine and the biosynthesis of pyrimidine, thereby regulating the synthesis of nucleotides during cell proliferation [160].

TERT
Telomerase reverse transcriptase (TERT) is a component of telomerase. Compared with most normal cells lacking telomerase activity, telomerase activity is up-regulated in many malignant tumors, including thyroid cancer, NB, etc., allowing cancer cells to replicate indefinitely [4,176]. Evidence shows that TERT can prevent cell cycle arrest and prevent cell apoptosis induced by poor culture conditions in vitro [177,178].
In NB, N-Myc binds to the typical E-box near the transcription start site of the TERT gene, and the up-regulation of MYCN is accompanied by a 10-to 20-fold increase in TERT expression, suggesting that TERT is a target gene of N-Myc [88].
In Burkitt's lymphoma, cells treated with BIBR (TERT inhibitor) showed changes in the cell cycle spectrum, with decreased cells in the G1 phase, the disappearance of cells in the G2/M phase, and a large accumulation of cells in the S phase. The inhibition of TERT leads to DNA damage, then ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases are phosphorylated after sensing DNA-damage response (DDR) signals, thus activating checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2), and p53 proteins, and further phosphorylating Cdc25A protein to induce cell cycle arrest and accumulation in the S phase [151].
In many cancer cell lines, the decrease in hTERT (human TERT) expression is related to the significant down-regulation of CCND1, and the high expression of hTERT significantly up-regulates the expression of CCND1. In addition, the transcriptional activity and nuclear localization of Cyclin D1 are also regulated by TRET, suggesting that TERT promotes cell cycle progression by regulating the key cell cycle regulator Cyclin D1 [152].

PBK
The PDZ-binding kinase/T-LAK-cell-originated protein kinase (PBK/TOPK) is a serinethreonine kinase involved in the formation of the spindle and the process of mitosis [171]. PBK is highly expressed in lymphoma, myeloma, and primary hematological tumors, which is closely related to the malignant potential of these tumors [179][180][181].
In NB, the expression of MYCN and PBK is positively correlated. Knocking down MYCN leads to a decrease in PBK mRNA and protein levels. Chromatin immunoprecipitation sequencing (ChIP-seq) data analysis showed that N-Myc bound to the PBK promoter and was accompanied by the acetylation of lysine 27 of histone H3 (H3K27Ac), indicating that N-Myc can promote the transcription process of PBK [168].
The expression of PBK is related to mitosis. During mitosis, PBK forms a complex with Cyclin B1-CDK1 and phosphorylates the protein regulator of cytokinesis 1 (PRC1) in a Cyclin B1-CDK1-dependent manner. PRC1 is a microtubule-binding protein that participates in the formation of the mitotic spindle and promotes cytokinesis [171].
In human colon cancer cells, PBK interacts with p53, which contains the DNA binding domain (DBD), thereby down-regulating the transactivation function of p53 [169]. The CKI p21 is a well-known transcription target of p53 and plays a crucial role in mediating growth arrest [172]. Upon silencing PBK, the promoter activity of p21 and its mRNA significantly increased. Experiments proved that the overexpression of PBK down-regulated the expression of p21 by reducing the recruitment of p53 to the p21 promoter [169]. After knocking out PBK, the number of cells in the G2/M phase increased significantly and cell growth slowed down, indicating that the knockdown of the PBK gene may lead to cell cycle arrest and inhibit cell proliferation [169]. In addition, the expression levels of many other p53 target genes are also significantly changed due to PBK gene knockdown, such as the G2/M phase-specific E3 ubiquitin-protein ligase (G2E3) [170], Dual specificity protein phosphatase 1 (DUSP1) [182], etc., which regulate cell proliferation by participating in cell cycle regulation [169].
In the SGO1 genome sequence, there are four E-boxes (1)(2)(3)(4) in the 4kb region upstream of the start codon of SGO1, and an E-box is located in the intron of SGO1 [161]. ChIP analysis showed that N-Myc was recruited in E-box1 and E-box2 upstream of SGO1. Overexpression of MYCN induced up-regulation of SGO1 expression [161]. In addition, when MYCN was down-regulated in IMR32 cells, Sgo1 protein levels decreased. These results indicate that N-Myc up-regulates the expression of SGO1 by combining with E-box1 and E-box2 [161].
In NB cells overexpressing MYCN, using shRNA to knock down SGO1 severely inhibited cell proliferation, induced DNA damage, and caused cells to accumulate in the G2/M phase. [161] In prostate cancer cells, the high expression of SGO1 promotes cell proliferation and cell cycle progression [162]. Flow cytometry results showed that If SGO1 was induced to overexpress, the ratio of G0/G1 phase in cells decreased, and the ratio of S and G2/M phase increased [162]. When SGO1 was knocked down, the ratio of the G0/G1 phase increased, and the ratio of the S phase and the G2/M phase decreased. At the same time, Western Blot experiments showed that the expression of Cyclin A, CDK2, and Cyclin D1 was significantly reduced [162].
Among lung cancer cells, overexpression or knockdown of shugoshin-like 1 (SGOL1) prevents the correct alignment of the chromosomes in the metaphase equatorial plate, resulting in a delay at the beginning of the anaphase of mitosis [163].

AURKB
The serine/threonine protein kinase AURKB is the catalytic subunit of the chromosome passenger complex. It regulates many aspects of mitosis, including spindle checkpoints, chromosome segregation, and cytokinesis [189]. Overexpression of AURKB in a variety of human cancers shows a tendency for high malignancy and is positively correlated with poor prognosis [190].
ChIP-Seq data indicated that N-Myc can bind to E-box in the promoter region of AURKB [191]. GO analysis showed that the induction of high levels of MYCN resulted in the up-regulation of AURKB expression [153].
AURKB plays a regulatory role in the transition from the G2 phase to cytokinesis [190]. AURKB moves along the kinetochore to the equatorial region after the beginning of the later period, which is necessary for chromosome separation and cytokinesis [192]. Experiments showed that the lack of Aurora B interfered with the cell cycle and prevented the cell from completing cytokinesis, thus forming a tetraploid with two centrosomes [190]. After knocking out AURKB, the phosphorylation of histone H3 was significantly reduced, causing chromosome aggregation obstacles [155]. After injection of the AURKB antibody, AURKB is inhibited, blocking chromosome separation, covering the spindle checkpoint, and disrupting microtubule dynamics in mitosis [156].
In clear cell renal cell carcinoma, inhibition of AURKB induced cell accumulation in the G2/M phase and led to the down-regulation of Cyclin B and Cdc2 [154]. It was found that the expression of Cdc25C decreased and the expression of p-Cdc2 (Tyr15) increased in the cells that silenced Aurora kinase. The accumulation of p-Cdc2 kept Cdc2 in an inactive state, causing the cells to be blocked in the G2/M phase [154].

E2F5
E2F transcription factor 5 (E2F5) is a member of the E2F family and plays an important role in regulating the proliferation of many types of tumors [164,193,194]. E2F5 can bind to pocket protein p107 or p130, inhibit tumor growth, and block cell cycle progression in the G1 phase [166,167].
ChIP experiments confirmed that N-Myc can directly bind to the E-box in the E2F5 gene promoter and up-regulate its expression [164]. Tetracycline induced the SHEP-Tet21N cell line to up-regulate the expression of MYCN, and Western Blot showed that the level of E2F5 protein increased [164].
A CCK-8 experiment found that the down-regulation of E2F5 significantly inhibited the proliferation of NB cells. When E2F5 was down-regulated by siRNA transfection, the proportion of cells in the G0/G1 phase increased, and the expression levels of CDK2 and CDK6 decreased [164]. It is suggested that E2F5 regulates the cell cycle progression of NB by affecting CDK2 and CDK6 [164]. In glioblastoma multiforme (GBM), silencing E2F5 effectively inhibited the proliferation of GBM cells, and the cell cycle was arrested in the G0/G1 phase [165] 3.7. TEAD4 The TEA domain transcription factor 4 (TEAD4), also known as transcriptional enhancer factor-3 (TEF-3), is a key molecule in the TEAD family. TEAD4 is up-regulated in a variety of tumors and is a potential tumor prognostic marker, especially in MYCN-amplified NB cells, and is a key component to drive their proliferation [173,195,196].
Studies have confirmed that N-Myc binds to the TEAD4 promoter region to up-regulate its expression. Silencing MYCN can down-regulate TEAD4 at the protein level. In addition, TEAD4 binds to the MYCN promoter. Therefore, they have a positive feedback loop [173].
In NB, silencing the TEAD4 gene induces significant aggregation of cells in the G0/G1 phase and a decrease in cells in the S phase. At the genetic level, multiple genes involved in cell cycle progression and DNA replication are inhibited, including cyclin-dependent kinases (CDK2, CDK1, CDC25B), cyclins (CCND1), DNA replication proliferating cell nuclear antigen (PCNA), minichromosome maintenance complex component 7 (MCM7), Cdc6), checkpoint kinases (CHEK1, CHEK2, WEE1), and others. The regulation of these genes by TEAD4 is not affected by TAZ/YAP in the Hippo classic pathway [173].

Discussion
Neuroblastoma is one of the serious threats to the life and health of children, especially infants and young children [197]. Because its onset is insidious and lacks specificity, it is difficult to detect and diagnose at an early stage, and it has often metastasized by the time it is diagnosed and the overall degree of malignancy is relatively high [198,199]. The risk classification of neuroblastoma is based on a comprehensive assessment of the child's age, MYCN gene amplification, and 11q chromosome aberrations [200,201]. Among them, the amplification and high expression of the MYCN gene are often positively correlated with the malignant progression and poor prognosis of various malignant tumors such as NB [202,203], receiving widespread concern.
In addition to the target genes mentioned in the text, the N-Myc downstream-regulated gene (NDRG) is a classic N-Myc downstream target gene (including NDRG1-4), and its expression is inhibited by N-Myc in NB [204]. The expression of NDRG1 is biphasic throughout the cell cycle, peaking in the G1 and G2/M phases and reduced to the lowest level in the S phase, indicating that it may play a potential role in the G0/G1 block by changing the expression of p21 Waf1/Cip1 and CDK1/4 [205]. However, although NDRG1 can up-regulate p21 Waf1/Cip1 in prostate and lung tumor cells, it does not affect the cell cycle and proliferation but instead inhibits cell migration [206]. However, the overexpression of NDRG1 has also been confirmed to reduce the expression of the Wnt response gene CCND1, thereby inhibiting the process of the cell cycle [207]. In addition, studies have shown that NDRG1 may be related to the attachment of mitotic spindles during shedding and the regulation of cytokinesis [208]. All of this evidence indicates that NDRG1 is bidirectional and cell-type-dependent in regulating the cell cycle, suggesting that it has multiple limitations as a target of anti-tumor drugs.
As for the gene of the Mre11/Rad50/NBS1 (MRN) complex, studies have shown that RAD50 is the target gene of N-Myc. Although there is no evidence that the other two are direct targets of N-Myc, both of them contain a CACGTG MYC binding sequence E-box, making them potential target genes of N-Myc [209]. MRN is the main sensor of a DNA double-strand break (DSB) [210]. When DNA damage is detected, it activates signaling molecules, such as protein kinase ATM, to trigger a wide range of DNA damage responses, including cell cycle arrest [211]. Studies showed that the knockdown of NBN or inhibition of Mre11 can effectively inhibit the proliferation of MYCN-amplified cells [209]. In summary, MRN as a potential downstream target of N-Myc may play a role in inhibiting tumor proliferation and could be used as a target for anti-tumor drugs, but studies on this have been inadequate.
This article summarizes and explains the mechanism by which N-Myc promotes tumor cell proliferation by regulating the cell cycle and cell division and provides new ideas for research on targeted drugs. At present, the research and development of targeted drugs for MYCN primarily has the following ideas: (1) Prevent the abnormal expression of MYCN: For example, JQ1, a small-molecule inhibitor of the bromodomain and the extraterminal domain (BET) protein, can effectively down-regulate MYCN gene transcription and inhibit the proliferation of MYCN-amplified NB cells [212]. In addition, studies have shown that the inhibitors of CDK7(THZ1) or CDK9(CYC065) can disrupt abnormal MYCNdriven transcription and inhibit MYCN gene transcription, becoming potential anti-cancer drugs [213,214]. (2) Furthermore, we can promote the degradation of N-Myc by acting on the stability regulation network of N-Myc [215]. For example, PLK1 can counteract the F-box and WD domain protein 7 (FBXW7)-mediated degradation of N-Myc by destroying the stability of FBXW7 ubiquitin ligase complex, thereby increasing the stability of N-Myc [122]. PLK1 inhibitor BI 2356 shows strong anti-tumor activity in NB cells in vitro and in vivo [216]. Moreover, in NB and SCLC, MYCN-amplified tumor cells are more sensitive to PLK1 inhibitor treatment than tumors with normal N-Myc copy numbers [122]. In addition, studies have shown that N-Myc may be methylated by PRMT5 to reduce its degradation by the proteasome [217]. Treatment with PRMT5 inhibitor EPZ015666 resulted in decreased MYC protein levels and medulloblastoma cell growth, indicating that PRMT5 inhibitors are potential treatments for MYCN-driven cancer [218]. (3) The formation of heterodimers between N-Myc and MAX could be inhibited, thereby inhibiting the effect of N-Myc on target genes [219]. The compound can compete with MAX for the binding site HLH-Zip on N-Myc, such as 10058-F4 [220] and MYCi361 [221], or reduce the formation of the MAX-N-Myc complex by stabilizing the MAX homodimer [222]. (4) Lastly, the target gene of N-Myc proteins could be inhibited, such as MDM2 [223]. All the small-molecule drugs mentioned above are summarized and sorted into Table 2, which will provide more information about compounds including the corresponding structure, target, cell-free assay, cell data, and clinical trials.
In this paper, we summarized ten target genes of N-Myc and their regulatory networks for the cell cycle (primarily Cyclins and CDK, but also involved in proteins that play key regulatory roles in cell cycle progression, including proteins at cell cycle checkpoints). However, at the same time, we also noted that another seven N-Myc target genes showed abnormal expression in neuroblastoma, and the abnormal expression (overexpression or inhibition) of these genes led to the abnormal cycle of various tumor cells, suggesting the existence of a potential network of action, which requires further study.
Moreover, given that MYNC amplification is a high-risk factor for many tumors, MYCN affects cell cycle progression by targeting downstream genes, and many antitumor drugs targeting MYCN downstream genes have been developed with great success, we believe that MYCN can also be used as a target for antitumor drug development, and has great research potential and application value.

Supplementary Materials:
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