The 2-OG-dependent dioxygenase AspH hydroxylates aspartate and asparagine residues in certain EGFDs of its substrates, in particular Notch homologues or Notch ligand homologues (4,6,18). The Notch signaling cascade is a highly conserved pathway that affects cell differentiation, proliferation and apoptosis by mediating cell-cell communication, which is essential for human growth and development (24,25). Mammals have four Notch receptors (Notch1-4) and two ligands [Delta-like and Jagged (JAG)] (26). Both the Notch ligands and the extracellular domain (ECD) of Notch receptors contain tandem EGF-like repeats (27–29). Under the condition of β-hydroxylase activity, AspH binding ligands and receptors in a ligand-dependent manner enhances the stability and interaction between Notch receptors and ligands, leading to conformational changes in Notch (26,30,31). This process makes Notch more sensitive to continuous cleavage by a disintegrin and metalloproteinase (ADAM; S2 cleavage) and by the multiprotein γ-secretase complex (S3 cleavage) (26). On the other hand, AspH promotes the cleavage of the γ-secretase complex by directly interacting with ADAM10/17, releasing the Notch intracellular domain, which enters the nucleus and recruits coactivator proteins from the mastermind-like 1 (MAML1) family, forming a Notch transcription activation complex with recombination signal binding protein Jκ (RBPJ), also known as CSL [CBF1-Su(H)-LAG1] (26,29). Subsequently, downstream Notch-responsive genes are activated, including hairy and enhancer of split-1 (HES1), hairy-related transcription factor-1 (HEY1), CD44, epithelial cell adhesion molecule, c-Myc, MMP2/9, cyclin D3 and proliferating cell nuclear antigen (Fig. 1) (29,32). It has been demonstrated that the Notch signaling pathway serves a role in regulating exosomes, which are transferred from mesenchymal cells to tumors to promote metastasis (33,34). The activation of the AspH-Notch axis induces MMP/ADAM-mediated exosomal synthesis and release, and the latter markedly enhances breast cancer cell extracellular matrix (ECM) degradation/remodeling, infiltration and metastasis (both in vitro and in vivo) (32). In addition, the structural and functional abnormalities of tumor blood vessels, combined with diffusion deterioration, lead to decreased oxygen levels in regions within solid tumors and induce the expression of stress response proteins, such as hypoxia-inducible factor-1α (HIF-1α) (35). Chen et al (36) demonstrated that HIF-1α activates Notch signaling by synergizing with the Notch coactivator MAML1 and subsequently increases both HES1 and HEY1 expression levels under hypoxia. In addition, as the upstream target gene of AspH, HIF-1α enters the nucleus and controls AspH expression at the transcriptional level (37). Upregulated AspH expression stimulates the translocation of Notch to the nucleus by binding to Notch ligands and receptors, consequently governing downstream target genes that mediate cell adhesion, including E-cadherin and tenascin C (30,36–38). This novel molecular mechanism for HIF-1α-AspH-Notch signaling may serve an important role in cancer invasion and metastasis (Fig. 1).

Several studies have indicated that the MAPK and PI3K signaling pathways are the most general events in various types of human cancer (39,40). The abnormal activation of these proteins affects numerous biological processes, including cell proliferation, differentiation, growth, survival, motility and metabolism (39–41). It has been demonstrated that insulin and insulin-like growth factor (IGF-1) stimulate the intrinsic tyrosine kinase activity of the IGF-1 receptor, subsequently activating the PI3K and MAPK signaling pathways and causing the expression of downstream target substrates, including AKT and ERK (42–44). de la Monte et al (45) reported that insulin and IGF-1 induce the phosphorylation and activation of the PI3K and MAPK cascades, which stimulate AspH expression and enhance cell motility in hepatocellular carcinoma. Furthermore, GSK3β, which is downstream of both the PI3K and MAPK signaling pathways, is phosphorylated (inhibition) at Ser9 by its upstream kinases AKT and p38 (46). However, high levels of AspH lead to decreased GSK3β phosphorylation, which delays tumor cell senescence and promotes tumor progression by interfering with the communication between GSK3β and upstream kinases (Fig. 2A) (47).

Compared with surgery, radiation and chemotherapy, immunotherapy has provided important benefits to patients with melanoma (48). The purpose of cancer immunotherapy is to promote tumor-specific T-cell responses. In the presence of major histocompatibility and CD28 co-stimulation, the T-cell receptor interacts with antigens to activate T cells, which migrate to tumors, upregulate the expression levels of immune checkpoints, such as cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed cell death 1, and produce cytokines such as IFN-γ, which leads to the expression of programmed cell death ligand 1 (PD-L1) on tumor cells (48). CTLA-4 and PD-L1 are negative regulators that inhibit T-cell activation and induce tumor cell immune escape (49,50). Therefore, numerous efforts have been devoted to the development of inhibitors targeting immune checkpoints, including ipilimumab and nivolumab; these antibodies promote antitumor CD8⁺ cytotoxic T lymphocyte (CTL) responses in patients with melanoma (51). In addition, CD4⁺ T cells promote both the effector and the memory functions of CTLs and enhance their antitumor responses (51). The AspH protein is exposed to the extracellular environment of tumor cells and can be recognized and attacked by the host immune system (52). AspH contains both HLA class I- and class II-limited epitopes, which stimulate AspH antigen-specific CD4⁺ and CD8⁺ T-cell responses in human and animal models to elicit antitumor effects (Fig. 3) (52). λ phage nanoparticles expressing human AspH-derived proteins and AspH protein-loaded dendritic cells (DCs) migrate from the blood to lymph nodes to activate antigen specific CD4⁺ and CD8⁺ T cells; subsequently, T helper (Th)1 and Th2 immune responses are induced to promote lymphocytic infiltration and widespread necrosis in tumors (52,53).

HCC is the primary hepatic malignancy, with the highest incidence (~75%) among liver cancer worldwide in 2019 (54). Although therapeutic efforts have improved over the last few decades, the mortality rate of HCC has increased by 2.8 and 3.4% per year in men and women, respectively (55). Therefore, there is an urgent need for new treatment methods and a deeper understanding of HCC. The relevance of AspH modification in HCC has been extensively studied, and several studies have revealed that AspH is highly expressed in HCC and is associated with cell proliferation, invasion and malignant transformation (1,11,31,56–58). AspH binding to GSK3β inhibits its phosphorylation and inactivation, and blocks the interactions with the upstream kinases AKT and p38 (47). Inhibition of AspH enzymatic activity promotes HCC cell senescence and therefore delays tumor progression by increasing the phosphorylation of GSK3β and p16 expression (Fig. 2B) (47). Additionally, a previous study has revealed that AspH promotes cell proliferation by upregulating cyclin D1 and c-Myc expression (59). MicroRNA (miR)-200a, an upstream target gene of AspH that is rarely detected in liver tumor tissues and cell lines, suppresses cyclin D1 and c-Myc expression by downregulating AspH expression (59). Another study has revealed that AspH expression can be upregulated by insulin and IGF-1 in HCC (45). Insulin and IGF-1 stimulated AspH expression by increasing the phosphorylation of MAPK, ERK and AKT to enhance cell motility and invasiveness (45). Malignant phenotypes, such as tumor cell proliferation, migration, invasion and metastasis of HCC, are partially due to the activation of insulin and IGF-1, which increases AspH expression and subsequently activates the Notch signaling cascade (23,30,31). In addition, in HCC cells treated with an SMI (MO-I-1100) of β-hydroxylase, the activation of Notch signaling was inhibited, and the abilities of cell migration, invasion and metastasis were decreased compared with in untreated counterparts (23). Decreased copy number and dysfunction of mitochondrial DNA (mtDNA) are associated with the malignant phenotypes of HCC (60). AspH upregulation can destroy the integrity of mtDNA by blocking histone H2A member X-mitochondrial transcription factor A signaling, resulting in abnormal mitochondrial membrane potential, decreased ATP generation and increased reactive oxygen species; however, these effects can be reversed using small interfering RNAs against AspH (60). AspH is distributed on the surface of tumor cells, which makes it a target for immunotherapy. AspH-loaded DCs inoculated into HCC tumor-bearing mice can significantly suppress tumor growth, prolong survival and delay recurrence following surgical resection (52). Furthermore, both in healthy donors and patients with HCC, compared with α-fetoprotein-loaded DCs, AspH-loaded DCs can stimulate the activation of antigen-specific CD4⁺ T cells and CD8⁺ CTLs, which are important to initiate antitumor immune responses (61–63).

CC accounted for 10–25% of primary liver tumors globally in 2011, with a poor prognosis due to a lack of early diagnosis and effective treatment (64,65). It has been demonstrated that AspH is highly expressed in CC, while AspH upregulation is not observed in normal tissues, non-neoplastic epithelial cells and stromal cells (1). Clinicopathologically, AspH upregulation promotes CC invasion, metastasis and poor prognosis (66). Northern blotting suggests that AspH expression is upregulated in CC to promote intrahepatic spread and metastasis, since the AspH protein enhances the sarcomatous change and epithelial-mesenchymal transition (EMT) of CC (67). Additionally, the activation of the Notch signaling pathway was detected in CC; furthermore, enhanced Notch signaling and upregulation of downstream target genes (such as HEY1 and HES1) were observed when wild-type (wt)-AspH was transfected into HEK293 cells (68). As a cycle regulatory protein, cyclin D1 upregulation is closely associated with the progression and prognosis of CC (69). Knocking down AspH significantly downregulated cyclin D1 expression; however, overexpression of Notch partially rescued cyclin D1 levels, suggesting that AspH promotes CC cell proliferation through Notch-mediated cyclin D1 expression (68). In addition, in in vitro experiments, AspH-loaded DCs recruited CD3⁺ lymphocytes in tumor tissues to inhibit intrahepatic CC development and metastasis (70). In a CC model, a large portion of BDEneu-C24 cells expressed the AspH protein, causing a concentrated collagen matrix reaction during tumor formation; however, CD3⁺ T cells can penetrate the matrix barrier and reduce or delay the growth of CC (70). Recently, it has been reported that AspH promotes the growth and progression of CC by regulating the phosphorylation (and therefore inactivation) of RB1 (71). As a cancer suppressor gene, RB1 serves a vital role in cell cycle progression from G₀/G₁ to S phase and cell senescence (72,73). AspH upregulation increases the protein-protein interaction between RB1 and cell cycle-associated proteins, which in turn results in enhanced phosphorylation of RB1 (71). In addition, this interaction can be suppressed by inhibitors of hydroxylase activity (71).

PC was the third leading cause of cancer-associated mortality in the USA in 2019, with the lowest 5-year relative survival rate (9%) among all other types of cancer (74). The β-hydroxylase activity of AspH was proven to boost the malignant phenotypes of PC cells, such as cell migration, 2D and 3D invasion, EMT, ECM degradation/remodeling, stemness, microsphere formation and metastasis; these phenotypes were specifically suppressed using an SMI (MO-I-1182) (75). Additionally, it has been revealed that in a patient-derived xenograft (PDX) murine model with spontaneous pulmonary metastasis of human pancreatic ductal adenocarcinoma (PDAC), AspH promotes primary tumor development and pulmonary metastasis; these harmful effects can also be blocked using an SMI (MO-I-1182) (76). On the other hand, the proto-oncogene SRC can be activated by AspH through direct interaction with ADAM12/15 (75). Furthermore, the highly expressed AspH-SRC axis is a marker of poor prognosis in PC due to angiogenesis, invadopodia formation and metastasis (75,77). AspH can promote PC growth by activating Notch signaling cascades (29,78). Mechanistically, the ECD of Notch receptors contains 36 consecutive EGF-like repeats for the β-hydroxylation of aspartate/asparagine (27,29). AspH directly stimulates Notch to upregulate downstream responsive target genes, including HES1 and HEY1 (29). In AspH-overexpressing PDAC cell lines, a human monoclonal antibody against AspH (SNS-622-DM1) exerts significant antitumor effects by facilitating tumor cell G₂/M phase accumulation and increasing cellular cleaved caspase 3 expression (79). Additionally, SNS-622-DM1 can inhibit tumor growth and pulmonary metastasis in a PDX murine model (79).

CRC is the fourth most deadly cancer, with ~900,000 deaths annually worldwide in 2019 (80). A bioinformatics analysis revealed that the mRNA and protein levels of AspH are upregulated in CRC compared with in normal tissues due to gene copy number variations and promoter demethylation (81). AspH accumulates at the invasive tumor margin, which may be associated with cell invasion and infiltration (81). It has been recently reported that Notch signaling recruits TGFβ-dependent neutrophils to drive CRC metastasis; this pathway has an important role in the tumor microenvironment and predicts a poor survival in patients with CRC (82). Notably, knocking down AspH or using specific SMIs (MO-I-1144) decreases Notch expression in CRC, inhibiting tumor development and metastasis (81).

Studies have revealed the presence of AspH gene amplification in invasive/advanced ductal carcinoma and AspH silencing in normal adult breast tissues (32,83). AspH upregulation activates the Notch signaling pathway, increases the synthesis/release of pro-oncogenic exosomes and subsequently enhances EMT, 2D and 3D invasion, stemness, angiogenesis and metastases in breast cancer; these malignant phenotypes are reversed using an SMI (MO-I-1182) (32). AspH stimulates the Notch cascade by directly interacting with Notch receptors, ligands (JAGs) or ADAM10/17 modulators (32). The AspH-Notch axis is essential for the progression and prognosis of breast cancer (32). In mouse models, high levels of AspH induced more aggressive tumors, characterized by rapid growth and extensive metastases (32). Notably, phage vaccination markedly decreased pulmonary metastasis and enhanced survival in the 4T1 breast cancer model (with AspH overexpression) (53). On the other hand, in estrogen receptor-positive breast cancer cells, the activation of MAPK and PI3K cascades upregulates AspH mRNA expression when tamoxifen sensitivity is decreased (84). Furthermore, upregulated AspH expression decreases the progression-free survival of patients with luminal B breast cancer who received adjuvant endocrine therapy (84). Therefore, endocrine sensitivity of endocrine-resistant breast cancer with high AspH expression may be restored by blocking the MAPK and PI3K signaling pathways (84).

GBM was the most common primary malignant brain tumor among adults worldwide in 2016 (85). Via analyzing whole genome alternative splicing events in 498 GBM cases, it was revealed that AspH expression is upregulated in GBM and is associated with the onset and progression of cancer (86). A previous study has demonstrated that protein levels of AspH and of the proliferation-associated protein Ki-67 are upregulated in more aggressive GBM cases compared with well differentiated cases (87). Furthermore, AspH knockdown or SMI (MO-I-1100, MO-I-400, MO-I-500 and MO-I-1151) treatment targeting hydroxylase activity decreases the viability and directional motility of GBM cells (87). Moreover, shorter progression-free survival and overall survival are associated with AspH upregulation and HIF-1α expression in patients with GBM, analyzed using immunohistochemistry (87). The Cancer Genome Atlas gene database revealed that AspH and HIF-1α were significantly upregulated in the mesenchymal subtype of GBM (87). This demonstrates that both AspH and HIF-1α may be involved in mesenchymal transformation and may subsequently induce aggressive and invasive phenotypes (87).

Similarly to the aforementioned types of cancer, modulation of AspH function serves a critical role in endometrial cancer (EC), neuroblastoma, non-small cell lung carcinoma (NSCLC) and gastric cancer (88–92). Compared with normal cell lines, AspH expression was upregulated in EC cell lines, while miR-135a expression was downregulated (88). Cell Counting Kit-8 and wound-healing assays revealed that cell proliferation and migration were decreased by miR-135a overexpression. Conversely, high levels of AspH led to increased cell proliferation and migration, and miR-135a overexpression decreased the luciferase activity of EC cells transfected with wt-AspH 3′-untranslated region (UTR) but not mutant-AspH 3′-UTR (88). AspH upregulation restored the inhibitory effects of miR-135a on EC cells (88). These observations suggest that miR-135a affects EC growth and invasion by regulating AspH levels (88). AspH expression was significantly increased in neuroblastoma cells compared with in CNS-derived primitive neuroectodermal tumor cells. Mechanistically, insulin and IGF-1 increased directional motility by inducing AspH expression (89). However, treatment with AKT, ERK or cyclin-dependent kinase 5 (CDK-5) inhibitors significantly decreased insulin- and IGF-1-stimulated AspH mRNA expression and motility (89). These results suggest that ERK, AKT and CDK-5 signaling may mediate insulin and IGF-1 regulation of AspH at the level of transcription (89). In addition, high expression levels of AspH significantly enhanced neuroblastoma Sy5y cell motility, while the inhibition of AspH by antisense oligodeoxynucleotides decreased the motility of Sy5y cells and enhanced the expression levels of p21/Waf1 and p16, indicating that AspH is involved in tumor invasion and metastasis (90). In NSCLC, FB50 immunohistochemical staining revealed a marked increase in AspH expression, particularly in squamous cell carcinoma (91). High levels of AspH immunoreactivity are associated with poor survival and prognosis in patients with NSCLC, and AspH upregulation may increase the potential for tumor invasiveness and metastatic spread due to alterations in cell shape and adhesion (91). Finally, as a truncated isoform of AspH, humbug expression has been reported to be upregulated in several gastric cancer cell lines, especially in highly aggressive cells (92). High expression levels of humbug increased the anchorage-independent cell proliferation capability according to a colony formation assay; additionally, Transwell migration assays revealed that overexpression of humbug can promote cell migration and invasion compared with control vector-transfected cells (92). Therefore, humbug may be a molecule that affects the development and progression of gastric cancer (92).