Netrin-1 in cancer: Potential biomarker and therapeutic target?

Abstract

Netrin-1, a laminin-related protein, is known to be involved in the nervous system development. Recently, Netrin-1’s involvement in other processes such as cell adhesion, motility, proliferation, and differentiation that are important for the development of epithelial tissues has been described. In addition, Netrin-1 and its receptors, deleted in colorectal cancer and uncoordinated-5 homolog, have been linked to apoptosis and angiogenesis. Since these properties are essential for tumor development, Netrin-1 and its receptors have been reported to promote tumorigenesis in many types of cancers. Here, we review the Netrin-1 mediated regulation of cancer, its potential use as a biomarker, and the targeting of the Netrin-1 pathway to treat cancers.

Introduction

Netrin-1 belongs to a family of laminin-related secreted proteins and has been shown to play a major role in the control of neuronal navigation during the development of the nervous system.^1,2 Netrin-1 is expressed outside the nervous system and regulates diverse processes in addition to neuronal navigation such as adhesion, motility, proliferation, and differentiation of cells that contribute to the development of epithelial tissues.^3–6 Netrin-1 acts through two main receptors: DCC (deleted in colorectal cancer) and UNC5H (uncoordinated-5 homolog), and interaction of Netrin-1 with these receptors regulates inflammation, angiogenesis, and apoptosis, which in turn regulates tumorigenesis.^3,5,6 Apart from its role in the central nervous system, Netrin-1 is involved in the regulation of cancers, cardiovascular diseases, and kidney diseases, among others.^5,7,8 In recent years, it has become apparent that Netrin-1 may also be involved in the underlying pathology of several multisystem diseases, making it an attractive potential therapeutic target.^7,8

The two main Netrin-1 receptors, DCC and UNC5H, belong to the family of dependence receptors (DRs) that share the ability to regulate apoptosis positively or negatively in the absence or presence of their ligands, respectively. Therefore, it is advantageous for a tumor cell to lose this DR activity.⁵ This advantage was previously described in various human cancers. The expression of Netrin-1 is markedly reduced or absent in ~50% of brain tumors and prostate cancer, while it is overexpressed in many other cancers.^9–13 Moreover, Netrin-1 levels are significantly increased in many cancer types and secreted into the circulation.⁶ Thus, the use of Netrin-1 as a biomarker is being discussed in many human cancers.

In this review, we first discuss the current knowledge on Netrin-1, its receptors, and interactions between them. Then, we summarize the recent progress in understanding the regulation of the Netrin-1 status in various cancers. Finally, we discuss the possibility of using Netrin-1 as a biomarker and therapeutic target for cancer.

The Netrin protein family

The name Netrin, which means “one who guides” in Sanskrit, is derived from the word “netr.”^14,15 Netrins are a family of extracellular proteins that control axonal and cellular migration in embryogenesis. Three secreted (Netrin-1, 3, and 4) and two membrane proteins (Netrin G1 and G2) have been identified in mammals so far. Secreted Netrins act through their receptors. Netrin receptors identified in mammals belong to the DCC family, the Down syndrome cell adhesion molecule (DSCAM) family, and the UNC5H family: UNC5A, UNC5B, UNC5C, and UNC5D (UNC5H1, UNC5H2, UNC5H3, and UNC5H4). Netrin Gs do not interact with these receptors but control synaptic interactions between neurons.^15,16

Netrin-1 and its receptors

Netrin-1 is a 640 amino acid laminin-related protein. It is coded by NTN1 gene. The main function of Netrin-1 is to control neuronal navigation during the development of the nervous system. However, Netrin-1 is not only expressed in the central nervous system and neuroepithelial cells found in the ventral portion of the spinal cord but also in other non-neuronal organs.^1,2,17 DCC is a 1447 amino acid transmembrane receptor encoded by DCC gene. UNC5A, UNC5B, UNC5C, and UNC5D are transmembrane proteins encoded by UNC5A, UNC5B, UNC5C, and UNC5D genes. These receptors are referred to as DRs. The most important feature of DRs is their ability to launch two separate signal pathways. In the presence of ligand, they activate cell survival, migration, and differentiation, while in the absence of ligand, they do not remain inactive but induce apoptosis. Therefore, cells containing these receptors depend on the presence of ligand in order to survive in the extracellular environment. Thus, Netrin-1 does not only act as a chemotropic factor but also provides survival to cells.^2,3,5,16 Schematic representation of Netrin-1, DCC, and UNC5H is shown in Figure 1.

Figure 1.

Structure of Netrin-1 and Netrin-1 dependence receptors: DCC and UNC5H. DCC is a type I transmembrane protein with an extracellular domain composed of four immunoglobulin-like domains, six fibronectin type III domains, followed by a single transmembrane spanning region, and a cytoplasmic region composed of three conserved domains, namely P1, P2, and P3. There is a special site in the intracellular domain called addiction dependence domain (ADD). UNC5H receptors contain two extracellular immunoglobulin-like domains and two thrombospondin type I domains, a transmembrane region, and intracellular domains containing a ZU-5 domain and a death domain. Netrin-1 serves as a ligand and has three epidermal growth factor domains related to laminin, a laminin N-terminal domain, and a positively charged carboxy (C)-terminal domain.

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Apoptosis and Netrin-1 receptors

Apoptosis is a form of programmed cell death organized by a cascade of proteolytic enzymes called caspases. Apoptosis can be initiated via the intrinsic pathway when the cells sense stress and release contents from the mitochondria or via the extrinsic pathway by the ligands binding to cell surface death receptors. The majority of these receptors are members of the tumor necrosis factor receptor superfamily (TNFRSF). Extracellular stimulants can be tumor necrosis factor (TNF), insulin like growth factor (IGF), radiation, and drugs. Receptors on the cell surface detect these signals and pass into the cell through the cell membrane. Sequential activation of caspases occurs and this plays a central role in the cell apoptosis. Caspases exist as inactive proenzymes that undergo proteolytic processing to form the active enzyme. The caspase proteolytic activity results in the production of apoptotic bodies which are phagocytosed by neighboring macrophages resulting in death.^18,19

The DCC and UNC5H family receptors are DRs. DRs improve survival by inducing apoptosis when detached from ligands, while they inhibit apoptosis when bound to their ligands^2,3 (Figure 2). There is a private area in the intracellular region of the DCC receptor called ADD. The deletion of this region is sufficient to abolish the pro-apoptotic feature of DCC. In the absence of Netrin-1, cleavage of this region by Caspase-3 stimulates other caspases and facilitates apoptosis. UNC5H is also cleaved by Caspase-3 like DCC and the separated region of UNC5H induces apoptosis^16,20,21 (Figure 3).

Figure 2.

Netrin-1 and its receptor DCC representing the dependence receptor concept resulting in apoptosis. Dependence receptors are inactive when bound to their ligand and induce a death signal when the ligand is absent. Here, when Netrin-1 is bound to its receptor DCC (deleted in colorectal cancer), DCC transduces a positive signal leading to survival. When Netrin-1 is absent DCC amplifies a signal for programmed cell death.

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Figure 3.

Mechanism of DCC and UNC5H mediated apoptosis. When Netrin-1 is absent, both DCC and UNC5H are cleaved by active caspase in their intracellular domain. There is a special site called addiction dependence domain (ADD) in the intracellular domain of DCC. Cleavage results in the release of ADD. DCC–ADD leads to the recruitment of Caspase-9. This interaction allows Caspase-9 activation and Caspase-3 cleavage, leading to Caspase-3-dependent cell death. In case of UNC5H, the caspase cleavage results in the release of whole intracellular domain which contains the ZU-5 and the death domain, leading to the death signal.

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Netrin-1-dependent anti-apoptotic signaling is not completely understood. It may involve two steps: first, blockage of the caspase-related cleavage in the intracellular region of the receptors and second, the induction of anti-apoptotic signaling pathways such as extracellular signal–regulated kinases (ERK1/2) and AKT.^20–22

DCC and UNC5H as tumor suppressors

The pro-apoptotic properties of the DCC and UNC5H receptors in the absence of Netrin-1 have led to the idea that they are tumor suppressor genes. Hedrick et al.²³ reported that DCC could be a tumor suppressor gene because it was deleted in the majority of colorectal cancers as well as in many other cancers. More recent findings also support a tumor suppressor role for DRs.¹⁶ In contrast, these receptors can stimulate oncogenic pathways or inhibit apoptosis in the presence of Netrin-1.^16,24 Two studies found that in presence of Netrin-1, DCC activates the small guanosine triphosphatases (GTPases), CDC42–RAC1, or MAPK1/3 pathway which results in cell transformation in both transfected cells and commissural neurons and neuroblastoma cells.^25,26 Other studies confirm that DCC induces apoptosis in the absence of Netrin-1, while Netrin-1 blocks this affect.²⁷ Several other studies have shown a role for UNC5H as DR.^3,28 But on the contrary, DCC and UNC5H are inactive in most cancers. Therefore, the apoptotic properties of these receptors are lost in these cancers, and downregulation of these receptors represents a selective advantage for tumor development.^11,16 More studies are required to fully demonstrate the role of receptor dependency in various types of cancers.

Netrin-1 and cancer: a potential biomarker?

Netrin-1 is considered as an anti-apoptotic survival factor.^15,16 It was shown that binding of Netrin-1 to UNC5B results in the inhibition of p53-related apoptosis due to the stabilization of expression of a functionally inactive p53.^16,28 In light of this evidence, Netrin-1 can be considered as an oncogene. If Netrin-1 expression increases in cancer, p53-related apoptosis becomes halted after Netrin-1 signals through its receptor.¹⁶ Increased Netrin-1 expression was found in many tumor tissues, and increased apoptosis due to the downregulation of Netrin-1 expression resulted in tumor regression.^11,12,29 In recent studies, increased expression of Netrin-1 in hepatocellular carcinoma (HCC) tissues and cell lines was observed, and it has been shown that PI3K (phosphoinositide 3-kinase)/AKT signaling was activated in HCC.^30,31 Generally, decreased Netrin-1expression was found in colorectal cancer; however, it has been recently observed that a small fraction (10%) of colorectal tumors shows Netrin-1 upregulation.³² In many studies, expression of Netrin-1 was found to be elevated in breast cancer, lung cancer,colorectal cancer, pancreas cancer and glioblastoma, but it was not investigated whether Netrin-1 was being secreted by tumor cells and released into the circulation.9,11,13,29,33,34 We recently demonstrated the increased levels of Netrin-1 in serum of patients with gastric and lung cancer and showed that Netrin-1 levels decreased with chemotherapy. However, we could not detect any correlation between Netrin-1 and survival.^35,36 In a study by Ramesh et al., Netrin-1 was found to be elevated in breast cancer, prostate cancer, liver cancer, renal cancer, meningioma, pituitary adenoma, and glioblastoma. In this study, tissue and plasma samples of 300 patients with different cancers were compared with tissue and plasma samples of a control group of 138 patients with benign diseases. Only, tissue samples of renal cancer were obtained and compared with control groups. Netrin-1 expression in renal cancer was increased as well as plasma levels. It was the first study showing that Netrin-1 was secreted into the systemic circulation in cancer patients and could be detected in circulation. In addition, it was shown that Netrin-1 could be used as a biomarker for early detection of cancer but that was not specific to one type of cancer.⁶

Many studies have shown decreased expression of DCC and UNC5H in several cancers.^5,11,16,37 It has been shown that DCC and UNC5H expression decreased particularly in colorectal cancer and this was the result of a gene methylation in DCC and UNC5H.^5,12,37,38 Two other studies also showed loss of expression of these receptors by methylation in gastric cancer.^39,40 However, the expression levels of Netrin-1 were not measured and compared with the expression levels of the receptors. Therefore, further preclinical and clinical studies are needed to address the significance of Netrin-1 as a biomarker in cancer.

Netrin-1 and angiogenesis

After the identification of the logic that networks of developing blood vessels and nerves follow a parallel trajectory, the effects of Netrin-1 on embryonic vasculogenesis and angiogenesis were investigated in several studies.^7,41–43 The effect of Netrin-1 on the endothelium was shown to be similar to vascular endothelial growth factor (VEGF) signaling, which is known to stimulate nitric oxide (NO) production resulting in the inhibition of the cysteine-dependent, aspartate-directed, proteases (caspases) that induce apoptosis.^44,45 Netrin-1 promotes NO production via an ERK1/2-dependent pathway, and Netrin-1 directed NO stimulation also needs DCC activation of ERK1/2.^45,46 NO also contributes to ERK1/2 activation resulting in a feed-forward cycle. NO then mediates Netrin-1-induced enhancement in endothelial cell growth and migration.⁴⁶ However, controversial results have been shown in other studies where Netrin-1 bound to the UNC5B expressed on the endothelial cells resulted in the inhibition of angiogenesis.^47,48 In addition, contradictory observations of the role of Netrin-1 have been reported, acting as a pro- or anti-angiogenic factor, although current data largely suggests that Netrin-1 functions in pro-angiogenesis.^{7,41–43,47,48}

A separate mode of action through which Netrin-1 enhances endothelial cell survival also involves the UNC5B receptor. In its unbound state, UNC5B exerts a pro-apoptotic effect on endothelial cells, as mentioned earlier. Netrin-1 promotes the survival of endothelial cells upon binding to UNC5B.⁴⁹ However, Netrin-1 engagement to UNC5B receptor also has been reported to have an inhibitory effect on endothelial cell migration, with a consequent anti-angiogenic action.⁴²

Tu et al.⁵⁰ recently clarified the dual action of Netrin-1 on angiogenesis. In their study, the in vitro stimulatory effect of Netrin-1 on endothelial cell proliferation, migration, and tube formation was mediated by the pro-angiogenic CD146 adhesion molecule, for which Netrin-1 displays a higher binding affinity than for the classical UNC5B receptor. Hence, low concentrations (50–200 ng/mL) of Netrin-1 preferentially stimulate CD146. CD146 is a co-receptor for the VEGF receptor, VEGFR2; thus, Netrin-1 stimulation of CD146 facilitates VEGF signaling leading to angiogenesis. UNC5B activation only occurs at high concentrations of Netrin-1 (1000–2000 ng/mL) leading to downstream anti-angiogenic effects.⁵⁰ This study highlights the important role of CD146 in Netrin-1-induced angiogenesis, although other factors are also likely to contribute, as Netrin-1 can continue to stimulate angiogenesis, albeit to a far lesser degree, in the absence of CD146.^7,50

Targeting the Netrin-1 in cancer

Although, there are contradictory results, Netrin-1 expression is upregulated while its receptors DCC and UNC5H are downregulated in many cancers, as previously described.^{5,6,11–13,29–37} In a recent study, Netrin-1 expression was increased in non–small cell lung cancer (NSCLC). They concluded that targeting the interaction between Netrin-1 and its DR(s) might represent a potential approach for treating NSCLC.¹³ However, Netrin-1 plasma levels in colon cancer were not elevated and the majority of colorectal cancer cases were associated with the loss of Netrin-1 receptors. Therefore, although it is not clear in the majority of the cancers whether Netrin-1 or its receptor should be targeted, it is reasonable to target these receptors in colorectal cancers. When the studies are taken into consideration that the level of Netrin-1 is significantly increased in various cancers as mentioned earlier, it is unclear whether a treatment based on inhibiting the Netrin-1 itself or other strategies based on preventing the relationship between Netrin-1 and its receptors will be suitable alternative targeted strategies. Additional studies will be needed to determine whether anti-cancer treatments based on Netrin-1 itself and its interaction with its receptors represent viable alternative therapeutic approaches. Some compounds are currently under preclinical investigation.

Angiogenesis is a rate-limiting step for cancer growth.⁵¹ Neovascularization both precedes and is vital for tumor progression and metastasis.^52,53 It has been shown that Netrin-1 induces angiogenesis. Stimulation of angiogenesis depends on NO stimulation, and NO stimulation requires ERK 1/2 and DCC.⁴⁵ This angiogenesis dependency of the cancer has been confirmed by the treatment of tumors with angiogenesis inhibitors in many studies.^54–57 Therefore, knowing the concept that tumor growth is angiogenesis dependent, inhibition of the Netrin-1 stimulated angiogenesis pathway can be an important perspective for therapeutic approaches. Preclinical and clinical studies should be planned to investigate the therapeutic potential of Netrin-1 pathway in angiogenesis.

Targeting the Netrin-1 is of great interest because of the following: (a) It is not specific to a single type of cancer. It targets the patients with different types of cancers; (b) Theoretically, it is not associated with significant toxicities; (c) Again, theoretically, it may be combined with other treatment modalities; and (d) The study of the Netrin-1 pathway may expand the list of DRs and tumor suppressors, particularly with today’s advanced molecular studies.

Conclusion

In addition to its main role in the nervous system during embryogenesis, Netrin-1 also takes part in the other systems that makes it an important protein in oncology. Although there are contradictory findings on the role of Netrin-1 and its receptors in apoptosis and angiogenesis, that are important for tumor death and tumor growth, these properties highlight the possible roles of Netrin-1 in cancer and offer reasonable therapeutic approaches. Whether inhibition or augmentation of Netrin-1, its receptors or modulation of the interaction between them will provide clinical benefit in different tumors remains to be determined. Further preclinical and clinical studies are needed to determine its role in cancer.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.