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Introduction

According to the International Agency for Research on Cancer in 2024, there were ~20 million new cases of cancer in 2022 and 9.7 million deaths from cancer. Cancer has emerged as a significant public health issue (1). Projections based on demographics indicate that the annual number of new cancer cases may rise to 35 million by 2050, representing a 77% increase from 2022 levels. The global scope and varied cancer profiles across different regions and levels of human development underscore the importance of enhancing targeted cancer control measures worldwide (1). Despite notable advancements in cancer treatment modalities such as in surgery (2), targeted therapy (3,4), personalized cancer vaccines (5), gene editing technology (6) and chemotherapy (7), patients with advanced cancer continue to face poor survival rates and higher mortality (8). This highlights the critical need for identifying biomarkers and potential regulatory mechanisms for early tumor detection.

The ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family, also known as the extracellular nucleotide pyrophosphatase/phosphodiesterase family, consists of seven members, ENPP1, ENPP2, ENPP3, ENPP4, ENPP5, ENPP6 and ENPP7, which regulate extracellular pyrophosphate levels and serve a key role in influencing cell motility, migration, angiogenesis and tumor cell invasion (9–11).

The ENPP1 gene, located on chromosome 6q23.2, was originally identified as plasma cell (PC)-1, a PC differentiation antigen, in 1970. Its enzymatic activity and function as a transmembrane protein have been well studied (12). In earlier studies, researchers took advantage of the unique hydrolytic properties of ENPP1 and reported extensively on the involvement of ENPP1 in the regulation of bone mineralization and calcification, and its important role in phosphate homeostasis (13,14). Moreover, ENPP1 mutations are also associated with generalized arterial calcification of infancy (GACI) or pseudoxanthoma elasticum (15,16). However, the mechanism of action of ENPP1 in cancer has not been well studied; for example, in breast cancer, the earliest study dates back to 2010, which was a bioinformatic study that initially mentioned that ENPP1 may act as a gene with high statistical significance in relation to breast cancer (17). Furthermore, in 2013 it was identified as a potential promoter of breast cancer bone metastasis (18). In 2011, Aerts et al (19) reported that ENPP1 expression was associated with the grading of astrocyte tumors. Additionally, in 2012, Huhn et al (20) predicted that the effect of ENPP1 on metabolic syndrome risk and colorectal cancer risk may be highly dependent on other environmental factors or modifiers. Therefore, it can be seen that the early studies of ENPP1 in tumors did not involve deep mechanisms or information about its application in the clinic. The findings from the present review indicate that, in the initial stages of research, the focus was on the physiological and pathological functions of ENPP1 in non-tumor diseases. However, in recent years, there has been a shift towards investigating the regulatory mechanisms of ENPP1 in tumor diseases and the development of ENPP1 inhibitors, with a growing number of preclinical models and clinical trials (21–24). Based on these foundations, a number of studies in recent years have reported the regulatory mechanisms of ENPP1 in tumors. For example, in breast cancer, products generated by ENPP1 were reported to enhance the expression of the haptoglobin protein (Hp), an inflammatory mediator that can cause bone marrow invasion and promote neutrophil extracellular traps (NET) formation (25). Moreover, in lung cancer, ENPP1 expression was reported to be upregulated, with dysregulated ENPP1 leading to increased malignancy in human lung cancer by inducing cancer stem cell (CSC) features and epithelial-mesenchymal transition (EMT)-like phenotypes (10). Downregulated exosome-associated gene ENPP1 was also identified as a novel lipid metabolism- and immune-related biomarker in hepatocellular carcinoma (26).

Therefore, the present review summarizes early and recent studies of ENPP1 in several types of cancers, as well as the regulatory mechanisms (Table I) and clinicopathological features (Table II) that provide new insights into the mechanisms of cancer development.

Table I. Functional characterization of ectonucleotide pyrophosphatase/phosphodiesterase 1 in several cancer types.

Table I.

Functional characterization of ectonucleotide pyrophosphatase/phosphodiesterase 1 in several cancer types.

First author/s, year	Cancer type	Expression	Function roles	Related genes	Role	(Refs.)
Lau et al, 2013	Breast cancer	Upregulated	Metastasis	-	Oncogenic	(18)
Takahashi et al, 2015	Breast cancer	Upregulated	Proliferation	miR-27b, ABCG2	Oncogenic	(61)
Kawaguchi et al, 2019	Breast cancer	Upregulated	-	-	Oncogenic	(62)
Ruiz-Fernandez de Cordoba et al, 2022	Breast cancer	Upregulated	Proliferation	Hp	Oncogenic	(25)
Goswami et al, 2022	Breast cancer	Upregulated	EMT, proliferation, metastasis	2′3′-CGAMP, Vimentin, N-cadherin, E-cadherin, TWIST1, IFN-β	Oncogenic	(63)
Zhang et al, 2023	Gallbladder cancer	Upregulated	-	-	Oncogenic	(83)
Yano et al, 2004	Gallbladder cancer	Upregulated	Differentiation, migration	-	Oncogenic	(81)
Bageritz and Goidts, 2014	Glioblastoma	Upregulated	Proliferation, cell cycle	E2F1	Oncogenic	(11)
Aerts et al, 2011	Glioblastoma	Upregulated	-	-	Oncogenic	(19)
Bageritz et al, 2014	Glioblastoma	Upregulated	Differentiation, proliferation, apoptosis, cell cycle	E2F1	Oncogenic	(76)
Li et al, 2022	Liver cancer	Downregulated	-	-	Non-oncogenic	(26)
Hu et al, 2019	Lung cancer	Upregulated	Tumor progression, proliferation, apoptosis, migration, EMT	ABCG2, SOX2, NANOG, CD44, TGF-β, E-cadherin, Vimentin	Oncogenic	(10)
Ma et al, 2024	Oral squamous cell carcinoma	Upregulated	Proliferation, apoptosis, autophagy	PCNA, cyclin D1, BAX, Cl-caspase3, caspase3, Bcl-2, LC3B I, LC3B II, SQSTM1, ATG5, p-PRKAA1, PRKAA1, p-ULK1, ULK1	Oncogenic	(93)
Wang et al, 2021	Ovarian carcinoma	Upregulated	Proliferation, apoptosis, migration, invasion, EMT	Caspase3, E-cadherin, N-cadherin, Vimentin, PCNA, MMP9	Oncogenic	(73)

[i] ABCG2, ATP binding cassette subfamily G member 2; ATG5, autophagy related 5; Bcl-2, B-cell lymphoma 2; BAX, Bcl-2-associated X protein; EMT, epithelial-mesenchymal transition; E2F1, E2F transcription factor 1; Hp, haptoglobin; IFN-β, type β interferon; LC3B, microtubule associated protein 1 light chain 3 β; miR-27b, microRNA 27b; MMP9, matrix metalloproteinase 9; NANOG, Nanog Homeobox; PCNA, proliferating cell nuclear antigen; PRKAA1, protein kinase AMP-activated catalytic subunit α1; SOX2, SRY-Box transcription factor 2; TGF-β, transforming growth factor β; ULK1, unc-51 like autophagy activating kinase 1.

Table II. Clinical significance of ectonucleotide pyrophosphatase/phosphodiesterase 1 in several cancer types.

Table II.

Clinical significance of ectonucleotide pyrophosphatase/phosphodiesterase 1 in several cancer types.

First author/s, year	Cancer type	Clinicopathologic features	(Refs.)
Takahashi et al, 2015	Breast cancer	Recurrence-free survival	(61)
Kawaguchi et al, 2019	Breast cancer	Recurrence-free survival	(62)
Ruiz-Fernandez de Cordoba et al, 2022	Breast cancer	Poor survival	(25)
Goswami et al, 2022	Breast cancer	Tumor volume	(63)
Aerts et al, 2011	Glioblastoma	WHO grade	(19)
Li et al, 2022	Liver cancer	Poor prognosis	(26)
Hu et al, 2019	Lung cancer	Tumor burden and tumor size	(10)
Ma et al, 2024	Oral squamous cell carcinoma	Tumor volume and tumor size	(93)
Wang et al, 2021	Ovarian carcinoma	Preoperative serum, volume of ascites, tumor maximum size, FIGO stage, differentiation grade and chemotherapy sensitivity	(73)

[i] FIGO, International Federation of Gynecology and Obstetrics; WHO, World Health Organization.

Structure of ENPP1

ENPP1 is a membrane-bound glycoprotein that contains two consecutive somatomedin B-like domains (SMB; SMB1-2) connected together. It is classified as a type II transmembrane protein and also includes a phosphodiesterase domain, a catalytic domain (CS) and a nuclease-like domain (NUC) at its C terminus. The structure of ENPP1 is shown in Fig. 1. These domains share a structural organization with ENPP2 and ENPP3. The biological functions of ENPP1 are mediated by its different domains, with the CSs and NUCs working together in mineralization, whilst the SMB1-2 serve a role in insulin signaling (27).

Figure 1. Structure of ectonucleotide pyrophosphatase/phosphodiesterase 1. NUC, nuclease-like domain; PDE, phosphodiesterase domain; SMB, somatomedin B-like domain; TM, transmembrane domain; CD, cytoplasmic domain.

Physiological and pathological functions of ENPP1

ENPP1 is involved in purine and adenosine metabolism

As early as 2006 it was discovered that ENPP1 is a phosphodiesterase that can hydrolyze nucleotides and can also regulate purinergic signaling pathways (28). Furthermore, a 2019 study reported that adenosine triphosphate (ATP), NAD+ and nucleic acids are abundant purines and that ATP and NAD+ are excitatory, adenosine has anti-inflammatory effects on immune cells, and that ENPP1 serves an important hydrolyzing role as a source of AMP (29). Another study reported that cardiac injury induced the expression of ENPP1, an enzyme that hydrolyzes extracellular ATP to form AMP. In response to AMP stimulation, cardiomyocytes release adenine and specific ribonucleosides, which either disrupt pyrimidine biosynthesis during the synthesis step of orotidine phosphate or rescue pyrimidine biosynthesis through gene targeting of the ENPP1/AMP pathway, thereby enhancing repair after cardiac injury (30). The aforementioned studies indicate the role of ENPP1 in purine metabolism and provide insights into the treatment of related diseases.

Pathological role of ENPP1 in the process of bone mineralization and calcification

Mineralization is an important part of the development of bones and teeth, during which organisms generate inorganic minerals through the regulation of biomolecules. Whilst ENPP1 is known for its role in regulating bone and soft tissue mineralization, it acts mainly through the generation of pyrophosphate, as pyrophosphate (PPi) is one of the inhibitors of hydroxyapatite. ENPP1 negatively regulates bone mineralization by hydrolyzing ATP to produce PPi (31–33). The functional role of ENPP1 in the regulation of mineralization was demonstrated in the ‘tiptoe’ walking mouse model (ttw/ttw) established in 1998, which was initially used to demonstrate an association between defective ENPP1 expression and altered bone mineralization (34). GACI is mainly caused by ENPP1 defects; therefore, inhibition of the ENPP1-PPi-Pi signaling axis could be a novel idea for the treatment of pathological calcification (35–37).

Role of ENPP1 in diabetes and obesity

ENPP1 is located on chromosome 6q23.2 and was identified as encoding a protein that inhibits insulin signaling (38). ENPP1 can interfere with insulin binding to the insulin receptor α subunit, affecting its activation and inhibiting insulin downstream signaling (39,40). It is also involved in the pathogenesis of type 2 diabetes mellitus (T2DM) (41). Moreover, several mutants of ENPP1 have been reported to be associated with childhood and adult obesity and to increase the risk of T2DM (42). These findings could provide a molecular basis for the physiological link between insulin resistance and obesity and offer a fresh perspective on the prevention of such diseases. However, to date, there are few studies on the relationship between ENPP1 and diabetes and obesity, and further work is required to elucidate its physiological role.

Mutations in ENPP1 are associated with Cole disease

Cole disease is a rare autosomal dominant disorder in which patients present with areas of hypopigmentation of the skin and thickening of the skin of the palms and feet. Notably, Cole disease has both an autosomal dominant and an autosomal recessive mode of inheritance, depending on the specific ENPP1 variant (43,44). A 2013 study using a genome-wide approach reported that mutations in ENPP1 were associated with Cole disease (44) and that the mutations occur in the structural domain of SMB2 (45). In conclusion, this mutational characterization of ENPP1 may provide genetic evidence for the treatment of similar skin diseases.

Pathological roles of ENPP1 in anti-aging

Klotho is a well-known anti-aging factor that regulates phosphate metabolism, and mice with mutations or defects in klotho exhibit a phenotype similar to human aging. Moreover, it has been reported that ENPP1 acts as an anti-aging factor under phosphate overload by regulating klotho expression, provided that ENPP1 is mutated. This result suggests that the ENPP1-klotho axis is required to prevent the aging phenotype under phosphate overload conditions (46–48).

ENPP1 in human cancers

A review of the literature revealed that ENPP1 has been the subject of relevant studies in a number of common human cancers. Accordingly, the present review summarizes the associations between ENPP1 and several human cancers in order of incidence, with reference to the latest global cancer statistics, released in 2024. The association of ENPP1 with cancer is presented in Fig. 2.

Figure 2. Molecular mechanisms of ENPP1 in different human cancers. ABCG2, ATP binding cassette subfamily G member 2; AMPK, adenosine 5′-monophosphate-activated protein kinase; BAX, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; EMT, epithelial-mesenchymal transition; ENPP1, ectonucleotide pyrophosphatase/phosphodiesterase 1; E2F1, E2F transcription factor 1; GSC, glioma stem cell; Hp, haptoglobin; miR-27b, microRNA 27b; MMP9, matrix metalloproteinase 9; NANOG, Nanog Homeobox; NET, neutrophil extracellular traps; PCNA, proliferating cell nuclear antigen; TGF-β, transforming growth factor-β; ULK1, unc-51 like autophagy activating kinase 1.

Regulatory mechanisms of ENPP1 in several cancers and current research status

ENPP1 and lung cancer

Lung cancer continues to be the primary cause of cancer-related deaths globally. The most recent global cancer statistics project ~2.5 million new cases and 1.8 million deaths in 2022, with lung cancer ranking first among men and second among women in terms of both incidence and mortality (1). Among the several subtypes of lung cancer, lung adenocarcinoma is the most prevalent subtype of non-small cell lung cancer, representing >40% of all cases (49). Whilst surgical resection is currently the preferred treatment for patients with early-stage lung cancer, they still face a significant risk of tumor recurrence even after complete resection (50). Therefore, the search for a novel and effective treatment strategy is crucial to enhance the prognosis and survival rates of these patients.

In 2019, Hu et al (10) first proposed that ENPP1 serves a role in the invasion and metastasis of lung cancer. Moreover, elevated levels of ENPP1 expression were reported in most human lung tumor tissues compared with adjacent normal lung tissue. The dysregulation of ENPP1 contributes to increased malignancy in human lung cancer by promoting CSC characteristics and EMT-like phenotypes. Additionally, Hu et al reported that downregulation of ENPP1 gene expression led to decreased levels of CSC markers and reversed the TGF-β-induced EMT phenotype, resulting in diminished cell migration, restoration of E-cadherin expression and suppression of vimentin-induced inhibition (10). A total of 3 years after the publication of this result, Han et al (51) analyzed data from the Gene Expression Omnibus database using weighted correlation network analysis, the Least Absolute Shrinkage and Selection Operator method and Cox proportional hazards regression, and reported that ENPP1 may serve as a potential predictor for the recurrence of early lung adenocarcinoma. However, whilst the therapeutic potential of ENPP1 in lung cancer has been established, the specific molecular mechanisms underlying its effects remain to be elucidated.

ENPP1 and breast cancer

Breast cancer is a prevalent form of cancer among women, as highlighted in the 2020 global cancer statistics. Surpassing lung cancer, breast cancer accounted for an ~2.3 million new cases (11.7%) in 2020, making it the most common cancer worldwide (52). In the latest 2022 global cancer statistics, breast cancer ranked second with 11.6% of new cancer cases, following lung cancer at 12.4% (1). Recent advancements in therapeutic methods and chemotherapy drugs have markedly improved the survival rates of patients with breast cancer. Despite these developments, distant metastasis (53) and acquired drug resistance (54) continue to be major factors contributing to the poor prognosis of patients with breast cancer.

Lau et al (18) reported an increase in ENPP1 expression in human breast cancer cells, MDA-MB-231 and MDA-MB-468, as well as that heightened ENPP1 expression enhanced the tumor-forming ability of MDA-MB-231 cells in bone. Later, Awolaran et al (55) identified ENPP1 as a factor expressed in breast cancer cells that mediates bone metastasis in breast cancer and also serves a role in cell proliferation and differentiation. Moreover, through RNA sequencing of the triple-negative/mesenchymal cell lines, MDA-MB-231 and MDA-MB-157, it was reported that ENPP1 is influenced by a circular RNA originating from the dedicator of cytokinesis 1 gene, leading to its upregulation (56). These findings are specific to circular RNA and offer novel perspectives on the involvement of RNA in breast cancer development and progression.

Compared with human mammary epithelial cells (HMEC), the breast cancer cell MDA-MB-231 exhibits overexpression of ENPP1 and CD73. The enzyme converts extracellular ATP released by cells into adenosine, which in turn stimulates the A3 receptor, promoting cell migration with frequent changes in direction. MDA-MB-231 cells, HMECs and neutrophils demonstrate distinct purinergic signaling mechanisms that regulate their motility patterns. The subcellular distribution of A3 adenosine receptors in MDA-MB-231 breast cancer cells serves a role in cell motility dysfunction (57). These findings suggest that targeting the purinergic signaling mechanism associated with ENPP1 could be a potential therapeutic strategy to interfere with breast cancer cell motility, thereby reducing the risk of cancer cell spread and metastasis (57). Additionally, it is hypothesized that the enzymatic function of ENPP1 serves a pivotal role in the promotion of breast tumor growth and metastasis by limiting the infiltration of adaptive immune cells, which ultimately leads to the enhancement of antitumor immunity. Furthermore, ENPP1 is postulated to serve a pivotal role in the tumor microenvironment. It functions as an innate immune checkpoint, inhibiting the activation of the cyclic GMP-AMP (cGAMP)-stimulator of interferon genes (STING) pathway and simultaneously enhancing the extracellular adenosine pathway (22). Therefore, the present review underscores the potential of ENPP1 as a target for cancer immunotherapy, offering new possibilities for enhancing immune checkpoint blockade therapeutic strategies (22).

The ‘immune hot’ microenvironment is defined by a significant presence of antitumor T cells that express markers of immune activation and exhaustion, including programmed cell death 1 (PD-1). By contrast, immune cold tumors do not have many antitumor immune cells and do not respond well to anti-PD-1 therapy (58). Through in vitro, in vivo and bioinformatics studies, Attalla et al (21) reported that human epidermal growth factor receptor (HER)2Δ16-dependent activation of ENPP1, an oncogenic splicing variant of the HER2, is associated with aggressive HER2+ breast cancer. This activation influences the immune microenvironment in HER2Δ16 tumors by modulating cytokine expression, reducing those that stimulate immune cells and increasing those with immunosuppressive effects. The presence of ENPP1 on the surface of HER2Δ16 tumor cells contributes to an immune-cold environment, suggesting a potential target for transforming immune-cold tumors into immune-hot tumors.

Schettini et al (59) reported that ENPP1 is a potentially safe and feasible new target for molecular and pathological subtypes of breast cancer antibody-drug conjugates and chimeric antigen receptor T-cell immunotherapy, and used the Human Protein Atlas to perform a comprehensive analysis of protein expression. Moreover, in a study characterizing gene expression profiles in estrogen receptor (ER)-positive breast cancer, differential expression of ENPP1 was reported between ERα+ and ERα-primary breast tumors. This finding was confirmed using reverse transcription-quantitative PCR and immunohistochemistry, indicating that ENPP1 may be a potential target for breast cancer treatment (17). A subsequent study also reported upregulation of ENPP1 in patients with ERα+ (60).

Furthermore, a study reported elevated levels of ENPP1 in patients with breast cancer, with ENPP1 being targeted by microRNA (miR)-27b to control its efflux activity by influencing the expression and surface localization of ATP binding cassette subfamily G member 2 (61). Additionally, in breast cancer, the ‘SP fraction’ is defined as CD44−high/CD24−low cells, aldehyde dehydrogenase-positive cells and side population cells. ENPP1 has been found to play a role in regulating SP in breast cancer, leading to partial drug resistance (61).

Kawaguchi et al (62) developed a new fluorescent probe, TG-mAMP, to assess the prognosis of malignant breast cancer by detecting ENPP1 activity with high sensitivity. This probe can also be used to screen ENPP1 inhibitors in chemical libraries, potentially leading to the discovery of effective and selective inhibitors. Therefore, the study of TG-mAMP as a fluorescent probe could provide a rapid and cost-effective method for predicting the prognosis of patients with malignant breast cancer. To date, efficient ENPP1 inhibitors have also been developed. A team of researchers has already reported that a selective and potent ENPP1 inhibitor, AVA-NP-695, regulates tumor metastasis by negatively modulating EMT in addition to its immunomodulatory effects. AVA-NP-695, as an immunomodulatory and anti-metastatic drug, provides a strong theoretical basis for the treatment of triple-negative breast cancer (TNBC) (63). Another study reported that an ENPP1 inhibitor could retard tumor growth in a mouse model of breast cancer (64).

ENPP1 and colon cancer

Colon cancer is a prevalent type of cancer in developed countries and a major contributor to cancer-related mortality. According to Cancer Outcomes 2022, there were ~1.93 million new cases of colon cancer, accounting for 9.6% of all cancer cases. Additionally, colon cancer causes ~900,000 deaths, representing 9.3% of cancer-related deaths (1). There is already evidence of a potential association between ENPP1 and an increased risk of colorectal cancer. An experimental study reported that specific alleles of polymorphisms in ENPP1 may be associated with an increased susceptibility to colorectal cancer among the Czech population. Furthermore, the impact of ENPP1 on the risk of both metabolic syndrome and colorectal cancer could be influenced by several environmental factors or modifiers (20). However, the molecular regulatory mechanism of ENPP1 in colon cancer has not been studied yet, to the best of our knowledge, so this may be a promising research direction.

ENPP1 and prostate cancer

Prostate cancer accounts for ~7% of male cancers worldwide, with >1.2 million new cases and >350,000 deaths annually, positioning it as a prominent cause of cancer-related mortality in men (65). Metastatic castration-resistant prostate cancer (mCRPC) is the most lethal form of prostate cancer (66). A recent study used RNA sequencing on a cohort of 60 patients and identified 14 gene clusters strongly associated with mCRPC bone metastasis, among which ENPP1 was highlighted. Subsequent Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed a connection between ENPP1 and the synthesis and secretion of parathyroid hormone. Despite these findings, experimental validation of the role of ENPP1 in prostate cancer remains pending, underscoring the need for further investigation (67).

ENPP1 and liver cancer

Liver hepatocellular carcinoma (LIHC) is a prevalent form of primary liver cancer, ranking sixth among the most common cancers globally (1). LIHC is characterized by a poor prognosis and high mortality rates, often being diagnosed in advanced stages. As a result, there is a critical need to identify biomarkers and therapeutic targets for improving LIHC prognosis (68). ENPP1, an exosome-related gene, was reported to be markedly downregulated in LIHC tissues based on comprehensive bioinformatics analysis from multiple databases, and patients with downregulated ENPP1 displayed a poor prognosis (26). Li et al (26) further assessed the co-expression network of ENPP1 to elucidate its role in potential signaling pathways such as fatty acid degradation and PPAR signaling pathways. Moreover, an immunological analysis revealed that ENPP1 may serve a role in several immune-related characteristics, and can function as an immunosuppressant, immunostimulant and chemokine. These findings contribute to a deeper understanding of ENPP1 in the pathogenesis and immune response of LIHC, offering new insights for ENPP1-related immunotherapy in clinical treatment. However, this is only a ‘dry’ experiment, and further research is required to verify this conclusion with ‘wet’ experiments.

ENPP1 and ovarian carcinoma

In 2022, it was estimated that >200,000 women would die from ovarian cancer (1). This type of cancer represents 2.5% of all malignant tumors in women but contributes to ~5% of all cancer-related deaths due to its high mortality rate. The primary emphasis in treating ovarian cancer lies in early detection and prevention (69).

Extracellular nucleotides and nucleosides serve a crucial role in regulating tissue homeostasis. ENPP1 is known to hydrolyze ATP and cleave several substrates and chemical bonds (70). It has been reported that ovarian cancer cells, specifically SKOV-3 cells, express ENPP1. Notably, the conversion of ADP to AMP serves as a control point for the SKOV-3 cell phenotype, indicating a potential mechanism for regulating cell behavior. This highlights the significance of extracellular purine ratio in the migration phenotype of ovarian cancer cells. ENPP1, as a key hydrolase, serves a vital role in this process (71).

In 2020, Zhang et al (72) used bioinformatics techniques to forecast genes related to tumor metabolism. The study also revealed a potential association between ENPP1 and the prognosis of patients with ovarian cancer. Additionally, they developed a network of metabolism-related genes, which included ENPP1. By performing further gene analysis, high-risk groups can be differentiated from low-risk groups and additional clinical insights could be elucidated. Based on these currently known theoretical foundations, a previous study reported that the expression of ENPP1 mRNA and protein in high-grade serous ovarian carcinoma was notably higher compared with normal ovarian tissue. Moreover, the study revealed that the stronger the expression of ENPP1, the more advanced the International Federation of Gynecology and Obstetrics stage, and the poorer the differentiation of tumor cells. However, the research did not establish an association between EMPP1 expression and chemosensitivity. By interfering with the expression of ENPP1, the study reported a marked reduction in the proliferation, metastasis and invasion of ovarian cancer cells. This suggests that ENPP1 could potentially serve as a target for molecular treatment of ovarian cancer (73).

ENPP1 and glioblastoma

Glioblastoma, the most common primary brain tumor and one of the deadliest human cancers, can develop anywhere in the central nervous system (74). With a 5-year survival rate of only ~10%, there is an urgent need for effective treatments (75).

Glioblastoma encompasses astrocytomas derived from astrocytes. In 2011, it was first proposed that glioblastoma could be graded based on ENPP1 expression levels (19). Subsequent studies have identified ENPP1 as a potential therapeutic target for glioblastoma (11,76). ENPP1 is frequently regarded as a proto-oncogene in this aggressive brain tumor and is highly expressed, underscoring its significant role (11,19). The knockdown of ENPP1 impairs cell proliferation and results in the accumulation of cells in the G1 phase of the cell cycle. Furthermore, ENPP1 functions upstream of E2F transcription factor 1 (E2F1), which may lead to a reduction in the transcriptional activity of E2F1 (11).

A subset of glioblastoma stem-like cells (GSCs) with characteristics similar to neural precursor cells has been identified as resistant to treatment, potentially contributing to the high recurrence rate of glioblastoma (77). Bageritz et al (76) reported that GSCs express high levels of ENPP1. By knocking out ENPP1 in cultured GSCs, they reported a decrease in stem cell-related genes, differentiation into astrocyte lineage, impairment of sphere formation and cell death. This led to an accumulation of cells in the G1/G0 cell cycle phase, increased sensitivity to chemotherapy and phenotypic changes attributed to reduced E2F1 transcriptional function. The expression of ENPP1 is crucial for maintaining undifferentiated proliferation state of GSCs in vitro. The present review did not find any clinically relevant experiments using the high expression of ENPP1 in GSCs, and no study has explored the molecular mechanism of ENPP1 in glioblastoma since 2014, to the best of our knowledge, which could be an entry point for the treatment of glioblastoma in the future.

ENPP1 and gallbladder cancer (GBC)

GBC is the most prevalent malignant tumor of the biliary tract, characterized by poor prognosis and a high likelihood of metastasis (78,79). It is estimated that in 2022, there would be ~130,000 new cases of GBC resulting in ~90,000 deaths (1). Currently, complete surgical resection remains a viable treatment option (80). However, the lack of distinct symptoms often leads to late-stage diagnosis, resulting in missed opportunities for optimal treatment (78). A comprehensive understanding of the molecular mechanisms underlying gallbladder cancer progression is crucial for improving treatment outcomes. Furthermore, early identification of effective prognostic biomarkers holds promise for enhancing the management of GBC.

Yano et al (81) first reported that ENPP1 is predominantly located on the apical cytoplasmic side of bile duct tumor cells in 2004, a finding that was corroborated through immunohistochemistry and western blot analysis. This finding indicates that ENPP1 may serve a role in the invasion of neoplastic bile duct carcinoma and holds promise as a potential tumor marker. In a previous study performed in 2021, researchers used transcriptome bioinformatics analysis to identify and validate early candidate diagnostic and prognostic RNA methylation-related genes for GBC. The study highlighted ENPP1 as a potential hub gene in the methylation pathway and bile metabolism-related pathways, with high expression levels of ENPP1 being markedly associated with poor prognosis in patients with GBC. This suggests that ENPP1 could serve as a novel biomarker for both early diagnosis and prognosis assessment in GBC (82).

A prospective study performed in 2023 demonstrated the potential development implications of the ENPP1 gene. Zhang et al (83) used ultrasensitive electrochemical cell sensors to accurately identify and detect circulating tumor cells (CTCs) and their resistance to chemotherapy in GBC. The researchers developed a three-layer silica-QDs/PEI@SiO2 electrochemical probe by encapsulating CdSe/ZnS quantum dots within silica nanoparticles. By incorporating anti-ENPP1 antibodies, the electrochemical probes were able to specifically label CTCs extracted from GBC, offering a novel approach for the creation of innovative chemotherapy regimens. However, no research team has performed a clinical trial to prove whether this treatment is feasible, to the best of our knowledge, so subsequent work could be focused on clinical validation and translation.

ENPP1 and oral squamous cell carcinoma (OSCC)

OSCC primarily affects the oral mucosal epithelium, representing ~90% of oral malignant tumors. Symptoms may include impaired taste and difficulty swallowing (84,85). In 2020, there were 377,713 reported cases of OSCC globally, with the majority occurring in Asian countries (86). Certain studies suggest that targeted oncogene sequencing could be a promising approach for the diagnosis, prevention and treatment of OSCC in the future (87–89). Notably, tumor progression often involves the accumulation of genetic mutations in tumor cells, leading to drug resistance (90,91). A study performed RNA-sequencing analysis on five SCC9-derived oral cancer cell lines and predicted that ENPP1 may serve as a potential biomarker and therapeutic target that interferes with the occurrence and progression of oral tongue squamous cell carcinoma (OTSCC). Analysis of 248 clinical data points during the trial revealed a marked association between the up- and down-regulation of ENPP1 and patient survival. Furthermore, the upregulation of the ENPP1 gene was associated with increased lethality, indicating that ENPP1 could be a promising molecular target for future OTSCC treatment studies. However, to date, there is no relevant data to substantiate the applicability of these findings in clinical studies, to the best of our knowledge (92). Based on this theoretical basis, Ma et al (93) later reported an association between ENPP1 and cytotoxic autophagy, leading to a novel treatment approach for OSCC. The study reported that reducing ENPP1 expression effectively suppressed OSCC cell growth and triggered apoptosis. Moreover, ENPP1 depletion activated the AMPK signaling pathway, resulting in increased Unc-51-like kinase 1 (ULK1) levels and AMPK phosphorylation in OSCC cells. This activation induced autophagy in OSCC cells, highlighting ENPP1 as a promising therapeutic target.

In conclusion, the expression and function of ENPP1 varies in different cancer types, and ENPP1 is upregulated in most tumors, such as lung cancer, breast cancer and glioblastoma. ENPP1 acts as an oncogene but is downregulated as an oncogene in hepatocellular carcinoma, and we consider that this may be related to the localization of the ENPP1 gene (6q23.2). However, according to bioinformatics predictions, ENPP1 is notably downregulated in liver cancer, which may be related to the pathogenesis of liver cancer. Therefore, further studies are needed to confirm these findings in order to elucidate more detailed regulatory mechanisms.

Signaling pathways associated with ENPP1

ENPP1 and cGAMP-STING pathway

In recent years, ENPP1 has been reported to serve an important role in the immune response to several stimuli through the cyclic-GMP-AMP synthase (cGAS)-STING pathway (94). In this pathway, cGAS senses and responds to self and pathogenic double-stranded DNA in the cytoplasm, catalyzing the conversion of GTP and ATP to cGAMP (95). cGAMP then binds to its endoplasmic reticulum membrane-localized STING and activates downstream transcription of interferon and other cytokines, triggering powerful antiviral (96) and anticancer defense mechanisms (97–99). ENPP1 is the main hydrolyzing enzyme of extracellular cGAMP, and the hydrolysis of cGAMP by ENPP1 can attenuate the cGAS-STING signaling pathway (100,101). Therefore, an ENPP1 inhibitor can be safely administered systemically to enhance the extracellular level of cGAMP and activate STING to achieve the anticancer effect (102,103). The signaling pathway diagram is presented in Fig. 3A.

Figure 3. Major signaling pathways associated with ENPP1. (A) ENPP1 and cGAMP-STING pathway. (B) ENPP1-Hp signaling pathway. (C) ENPP1-E2F1 signaling pathway. (D) ENPP1-AMPK-ULK1-cell autophagy signaling pathway. AMP, adenosine monophosphate; AMPK, adenosine 5′-monophosphate-activated protein kinase; ATP, adenosine triphosphate; BAX, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; CD73, 5′-nucleotidase ecto; cGAMP, cyclic GMP-AMP; cGAS, cyclic-GMP-AMP synthase; CTC, circulating tumor cell; ENPP1, ectonucleotide pyrophosphatase/phosphodiesterase 1; E2F1, E2F transcription factor 1; GTP, guanosine triphosphate; Hp, haptoglobin; NET, neutrophil extracellular traps; PMN-MDSC, polymorphonuclear myeloid derived suppressor cells; STING, stimulator of interferon genes; ULK1, unc-51 like autophagy activating kinase 1. This figure was created using Figdraw 2.0 (www.figdraw.com).

ENPP1-Hp signaling pathway

Locoregional failure (LRF) following surgery and post-irradiation in patients with breast cancer is associated with a poor prognosis (104). A recent study reported a strong association between ENPP1 and LRF, suggesting that ENPP1-overexpressing CTCs serve a role in promoting recurrence through self-seeding mechanisms. Whilst first ENPP1-generated adenosinergic metabolites enhance the expression of Hp, an inflammatory mediator that can cause bone marrow invasion and promote NET formation, researchers have identified an unexpected mechanism of the ENPP1-Hp signaling axis, namely, that ENPP1-highly-expressing CTCs have an increased fitness for homing and colonization of recurrent tumor beds with the recruitment of polymorphonuclear myeloid derived suppressor cells capacity. This infiltration of an immunosuppressed myeloid subpopulation is triggered by Hp tumor secretion via the ENPP1-water metabolite through an autocrine/paracrine loop (25). At present, no role has been identified for the ENPP1-Hp signaling pathway to serve in other tumor types, to the best of our knowledge, which needs to be further explored. The signaling pathway diagram is presented in Fig. 3B.

ENPP1-E2F1 signaling pathway

Knockdown of ENPP1 affects the homeostasis of the intracellular nucleotide pool, and this dysregulation may be attributed to the enzymatic activity of ENPP1 in the extracellular gap, leading to a reduction in the transcriptional function of the cell cycle regulator, E2F1. Notably, ENPP1 knockdown has been reported to impair cell proliferation and affect cell cycle progression (11). The signaling pathway diagram is presented in Fig. 3C.

ENPP1-AMPK-ULK1-cell autophagy signaling pathway

In this pathway, downregulation of ENPP1 leads to activation of AMPK, which in turn increases the expression and phosphorylation of ULK1, leading to cytotoxic autophagy and tumor growth inhibition. In recent years, only one study has reported a role for this pathway in oral squamous carcinoma, to the best of our knowledge, and there have been no studies related to this pathway in other common human cancers (93). The signaling pathway is presented in Fig. 3D.

Prospects of ENPP1 in clinical diagnosis and treatment of tumors

The present review identified almost no trials investigating ENPP1-related tumor therapeutic drugs, and there are no examples of using ENPP1 features to diagnose or treat cancer, to the best of our knowledge. However, the present review found that between 2019 and 2024, several novel inhibitors of ENPP1 have been in development for cancer immunotherapy, such as 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one, 3,4-dihydropyrido[2,3-d]pyrimidin-2(1H)-one, pyrrolopyrimidine and pyrrolopyridine derivatives and phthalazinone analogues (23,24,105–109). However, these inhibitors are currently only being tested in preclinical models and are rarely, if ever, actually used in clinical trials (106,107,110). Of these, there is only one ongoing clinical trial of an oral, potent, selective small molecule inhibitor of ENPP1 called RBS2418 (Riboscience), which has the potential to activate an antitumor innate immune response leading to an antitumor response in adult patients with advanced or metastatic tumors, and the clinical results have not demonstrated toxicity with increasing doses (111). Although only one clinical trial is currently underway, clinical trials with several ENPP1 inhibitors in combination with other therapies may be performed (111). In 2024, Sun et al (24) reported the use of an ENPP1 inhibitor in a 4T1 mouse model of TNBC and demonstrated in vivo efficacy. These experimental data provide a promising lead compound for cancer immunotherapy. The development of a large number of inhibitors indicates the potential of ENPP1 as a cancer therapeutic marker. The mechanism of ENPP1 in tumors has not yet been fully explored, therefore the elucidation of the inhibitors may begin as a starting point to gain a deeper understanding of the antitumor mechanism of ENPP1 inhibitors to perform more clinically meaningful trials and provide more theoretical basis for earlier treatment of cancer.

Discussion and conclusions

The majority of initial studies have concentrated on the distinctive hydrolytic characteristics of ENPP1. These properties were utilized not only to regulate purinergic signaling (28), but also to facilitate cardiac injury repair by regulating the ENPP1/AMP axis (30). Many studies have also reported the pathological role of ENPP1 in bone mineralization and calcification, which can be demonstrated by the classic model of ‘tiptoe’ walking mouse (ttw/ttw) (34), and the inhibition of the ENPP1-PPi-Pi signaling axis may be a new idea for the treatment of pathological calcification (35–37). The association between ENPP1 and diabetes and obesity is also strong, as ENPP1 has been identified as a protein that inhibits signaling downstream of insulin (38), and is also involved in the pathogenesis of T2DM (41). Several of its mutants have been implicated in obesity and Cole disease, but further study of the mechanisms involved is needed to better pinpoint the treatment of such diseases (42,44). A notable function of ENPP1 is that it regulates the expression of the anti-aging factor klotho and prevents aging by acting on the ENPP1-klotho axis (48). These several physiological and pathological functions could provide new ideas for the treatment of early non-tumor diseases.

In recent years, the emerging ENPP family has been identified as a potential proto-oncogene or tumor suppressor gene. These ENPPs serve a crucial role in the occurrence and development of several diseases and tumors, as highlighted in several studies (61,112–116). ENPP1, the most comprehensively studied member of the ENPP family, is widely expressed in several cancers including breast cancer, gallbladder cancer, glioblastoma and lung cancer (Table I). Conversely, Li et al (26) reported the downregulation of ENPP1 expression in liver cancer tissues, although this finding requires further validation through additional experiments.

Upregulation and downregulation of ENPP1 have been associated with several clinicopathological characteristics and prognosis, including recurrence-free survival, tumor volume, tumor size and World Health Organization grade (61–63,73,93). Additionally, ENPP1 severs a role in processes such as cell proliferation, migration, apoptosis, EMT and metastasis (10,18,63,73). Mechanistically, ENPP1 can act as a direct target gene of specific miRs, such as miR-27b, influencing downstream signaling pathways (61). It also impacts the function of transcription factor like E2F1, which in turn affects cell cycle, proliferation, GSC phenotype, and regulates tumor cell development (11,117). Knocking down ENPP1 has been reported to affect processes such as proliferation and EMT-related phenotypes (10,73). Based on these identified regulatory mechanisms, the present review summarized four major ENPP1-related signaling pathways in tumors, namely ENPP1 and cGAMP-STING pathway, the ENPP1-Hp signaling pathway, the ENPP1-E2F1 signaling pathway and the ENPP1-AMPK-ULK1-cell autophagy signaling pathway. Among them, the ENPP1 and cGAMP-STING pathway has been the most widely studied and applied, and researchers have elucidated inhibitors for cancer immunotherapy based on this pathway (118). The other three have only been found to be studied in breast cancer, glioblastoma and OSCC, and the research is highly limited (11,25,93). Therefore, these identified regulatory mechanisms could be used as a basis to fully explore the role of these signaling pathways in other cancers.

Several ENPP1 inhibitors have been developed, including the ENPP1 inhibitor AVA-NP-695 (63) which is specifically targeted at breast cancer, as well as other types of inhibitors such as nucleotide-based and non-nucleotide-based inhibitors (23,24,105,107,118,119). Despite progress in the discovery of these inhibitors, the diversity of their chemical structures and the efficacy of the drugs are not yet ideal, and their application in clinical trials has not been verified.

The molecular mechanism of ENPP1 in several cancers, including gallbladder and liver cancer, remains poorly understood. Clarifying the precise molecular pathway of ENPP1 would enhance the understanding of its involvement in cancer advancement, offering valuable clinical perspectives for tumor diagnosis and treatment.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Youth Natural Science Foundation of Shandong Province (grant nos. ZR2021QH367 and ZR2022QH146) and the Shandong Students' Platform for innovation and entrepreneurship training program (grant no. X2024265).

Availability of data and materials

Not applicable.

Authors' contributions

ZY, FP and LX conceptualized the study. LZ, YZ, XD, RL and KZ wrote the manuscript. XD, RL and YT designed the figures and tables. LZ, YT and FP revised and submitted the manuscript. Data authentication is not applicable. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

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References