NDPK-A, encoded by nm23-H1 (also known as NME1) was the first metastasis suppressor discovered. Much of the attention has been focused on the metastasis-suppressing role of NDPK-A in human tumors, including breast carcinoma and melanoma. However, compelling evidence points to a metastasis-promoting role of NDPK-A in certain tumors such as neuroblastoma and lymphoma. To balance attention on this contrariety of NDPK-A in different cancer types, this review addresses the metastasis-promoting role of NDPK-A in neuroblastoma. Neuroblastoma is an embryonic tumor, arising from neural crest cells that fail to differentiate into the sympathetic nervous system. We summarize and discuss nm23-H1 genetics and the prognosis of neuroblastoma, structural and functional changes associated with the S120G mutation of NDPK-A, as well as the evidence supporting the role of NDPK-A as a metastasis promoter. Also discussed are the NDPK-A relevant molecular determinants of neuroblastoma metastasis, and metastasis-relevant neural crest development. Because of NDPK-A’s dichotomous role in tumor metastasis as both a suppressor and a promoter, tumor genome/exome profiles are necessary to identify the molecular drivers of metastasis in the NDPK-A network for developing tumor-specific therapies.
Metastasis remains as the major cause of death in cancer patients, accounting for ~90% of cancer mortality. To metastasize, tumor cells need to successfully complete every step in the metastatic cascade, including detachment from the primary tumor, migration and invasion to local tissues, survival in the circulatory and lymphatic systems, and colonization at a distal organ(s) of the human body.
The tumor metastasis field has greatly advanced since the discovery, three decades ago, of the first metastasis suppressor gene, nm23-H1 (also known as NME1).1 The nm23-H1 gene encodes nucleoside diphosphate kinase A (NDPK-A, also termed as NM23-H1 and NME1),2 which belongs to the human NDPK family, currently consisting of ten members. Much of the attention has focused on the metastasis-suppressing role of NDPK-A in human tumors including breast carcinoma and melanoma. However, compelling evidence points to an opposite role for NDPK-A as a metastasis promoter in certain tumor types, such as neuroblastoma and lymphoma. To clarify this dichotomous trait of NDPK-A, this review will address the metastasis-promoting role of NDPK-A in neuroblastoma.
THE CLINICAL RELEVANCE OF NDPK-A TO TUMOR METASTATSIS
After a seminal report by Steeg et al,1 the clinical relevance of NDPK-A in tumor metastasis has been extensively studied. Results from most of these studies are summarized in Tables 1 and 2. A negative correlation between the protein and/or RNA levels of NDPK-A and metastatic potential is displayed by many cancer types, including breast, head and neck, liver and ovarian cancers (Tables 1 and 2). This negative correlation suggests a metastasis-suppressing role for NDPK-A (see the review by Steeg in this issue). For colorectal, gastric and lung cancers, however, the role of NDPK-A in tumor metastasis remains uncertain because of contradictory correlations (Tables 1 and 2). Conversely, a positive correlation between the protein and/or RNA levels of NDPK-A and metastatic potential occurs in neuroblastoma and lymphoma, suggesting a metastasis-promoting role of NDPK-A (Tables 1 and 2).
The dichotomous role of NDPK-A in tumor metastasis is likely due to the unique genetic makeup of different human cancer types. In addition to the different molecules and pathways affected, pediatric tumors such as neuroblastoma generally contain fewer mutations than adult tumors such as breast carcinoma, which display 10–20 and 25–130 non-synonymous mutations per tumor, respectively.3, 4 To address this contrariety of NDPK-A, we focus on its metastasis-promoting role in neuroblastoma by starting with the nm23-H1 genetics unique to this disease.
NM23-H1 GENETICS AND PROGNOSIS OF NEUROBLASTOMA
Neuroblastoma is the most common extracranial tumor of early childhood, accounting for 7% of all pediatric cancers.5 Neuroblastoma arises from multipotent neural crest cells that fail to differentiate into the sympathetic nervous system.6 Based on the International Neuroblastoma Staging System (INSS), limited stages (1 and 2) and advanced stages (3 and 4) of neuroblastoma are referred to as localized and metastatic tumors, respectively.7 The long-term survival rate for advanced stages of neuroblastoma patients is 40–50%.5
The genetics of nm23-H1 in neuroblastoma is more complicated than that in other tumors. An increased nm23-H1 copy number has been reported in 14% (13 of 95) to 23% (7 of 31) of neuroblastoma patients.8, 9 The nm23-H1 gene is mapped to chromosome 17q21.3.10 An increased nm23-H1 copy number therefore could be due to the gain of a chromosomal segment, 17q21-qter, which occurs in 54–65% of neuroblastoma patients and is associated with poor clinical outcomes.11, 12, 13
In addition to an increased gene copy number, high levels of nm23-H1 RNA and/or NDPK-A protein also occur in advanced neuroblastomas, and which are associated with poor prognosis.8, 9, 14, 15, 16 A high level of NDPK-A can occur in advanced neuroblastomas with or without MYCN amplification.14 MYCN amplification is a frequent genetic alteration, occurring in ~20% of patients with advanced neuroblastoma.6 The ability of MYCN to upregulate nm23-H1 expression17 can contribute to a high NDPK-A level in this subset of neuroblastomas. Intriguingly, neuroblastoma patients with MYCN amplification display a higher serum NDPK-A level than those without MYCN amplification.18 Serum NDPK-A suggests a secreted form, similar to that reported in myeloid leukemia.19
Although nm23-H1 mutations are rare, a serine 120glycine (S120G) mutation of NDPK-A has been reported in 21% (6 of 28) of advanced neuroblastomas, but not in any of 22 limited-stage tumors.20 This S120G mutation appears to be specific to neuroblastoma as it was not detected in 26 breast carcinoma patients nor in 17 patients with acute leukemia.20 This mutation can be inherited or occur somatically, and also can occur with or without nm23-H1 amplification (3–10 copies).20 Moreover, the S120G mutation of NDPK-A can arise in advanced neuroblastomas with or without MYCN amplification.20 This genetic heterogeneity likely complicates the interpretation of NDPK-A’s role in tumor metastasis.
On the basis of a differential expression of genes between favorable and unfavorable (ie, advanced) neuroblastomas, NME1 (also known as nm23-H1), CHD5 and PAFAH1B1 genes are proposed as a prognostic signature for risk stratification of neuroblastoma patients.21
NDPK-A OR NDPK-AS120G ENHANCES NEUROBLASTOMA CELL INVASIVENESS
Metastasis-associated cellular processes include decreased cell adhesion as well as increased cell survival, migration, invasion, and colonization. For simplicity here, these cellular processes are collectively termed cell invasiveness. It is noteworthy that cell proliferation and death are generally considered as tumorigenesis- and not metastasis-associated processes.
For NDPK-A to be a bona fide metastasis promoter in neuroblastoma, it is expected to increase the invasiveness but not the proliferation of neuroblastoma cells. This is indeed the case for human neuroblastoma NB69 cells that express ectopic NDPK-A or NDPK-AS120G.22 NDPK-A or NDPK-AS120G readily increases the invasiveness of NB69 cells, as measured by serum-independent survival, cloning efficiency, cell migration, and colony formation on soft agar.22 On the other hand, ectopically expressed NDPK-A or NDPK-AS120G does not affect the proliferation of NB69 cells under normal growth conditions.22 A similar migration-enhancing effect of NDPK-A or NDPK-AS120G is also observed in another human neuroblastoma cell line, SH-SY5Y (unpublished data). These two neuroblastoma cell lines do not exhibit MYCN amplification, which therefore excludes the possibility of interference by MYCN in the cell invasiveness-enhancing role of NDPK-A.
Compared with the wild type, ectopically expressed NDPK-AS120G level is lower but more potent in enhancing the invasiveness of NB6922 and SH-SY5Y cells (unpublished data). A lower level of NDPK-AS120G is not due to protein instability because a similar half-life is observed between the mutant and its wild type.23 This indicates that S120G may be a gain-of-function mutation.
A gain-of-function for S120G mutation is further observed in human cancer cell lines, in which NDPK-A behaves as a metastasis suppressor. NDPK-AS120G increases, whereas the wild type inhibits, the migration of human breast cancer MDA-MB-435 cells.24 In human prostate carcinoma DU145 cells, the S120G mutation abrogates the ability of NDPK-A to inhibit cell colonization and invasion.25 It seems reasonable to speculate that the genetic background unique to neuroblastoma, breast, and prostate carcinomas might dictate the role of wild-type NDPK-A in cell invasiveness. However, an apparent gain-of-function of S120G mutation renders NDPK-A with a better ability to enhance cell invasiveness regardless of tumor origins.
NDPK-A OR NDPK-AS120G PROMOTES NEUROBLASTOMA METASTASIS
Approximately 50% of human neuroblastomas originate from the adrenal gland,26 which serves as an ideal orthotopic site for a xenograft animal model of neuroblastoma metastasis. A fluorescent orthotopic xenograft model developed in SCID mice not only recapitulates human neuroblastoma, but also allows sensitive detection of GFP-labeled primary and metastatic tumors in mice.27 In this orthotopic xenograft model, NDPK-A- or NDPK-AS120G-expressing NB69 cells increase both the incidence and colonization of neuroblastoma metastasis in animal lungs without significantly affecting primary tumor development.22 Compared with the wild-type, NDPK-AS120G is more effective in promoting neuroblastoma metastasis in mice, consistent with their abilities in cell invasiveness.22 The lymphatic system appears to be one route for neuroblastoma cell dissemination because of accumulation of GFP-labeled NB69 cells in the inguinal lymph node of xenograft mice.27
As the xenograft mouse model is difficult to use for monitoring the behaviors of moving tumor cells, a xenograft zebrafish model has been developed, which is able to show that NDPK-A or NDPK-AS120G enhances the ability of NB69 cells to extravasate the fish tail vein (unpublished data). The extravasation-enhancing ability in xenograft zebrafish is consistent with the metastasis-promoting ability of NDPK-A or NDPK-AS120G in xenograft mice.22 Extravasation is essential for migrating tumor cells to gain access to other organs, an end point that is difficult to measure in the xenograft mouse model. Because of economic, physiological, and real-time observational advantages of zebrafish, this xenograft model will facilitate mechanistic and therapeutic studies of extravasation regulated by NDPK-A.
STRUCTURAL AND FUNCTIONAL CHANGES OF NDPK-AS120G
The molecular mechanism by which NDPK-A or NDPK-AS120G contributes to neuroblastoma metastasis remains unknown. Nevertheless, this mechanism is likely associated with S120G-associated structural and functional changes. Phosphotransferase activity is a well-established function of NDPK, including NDPK-A.28 Histidine 118 (H118) is an active site, and the H118-phosphorylated intermediate is essential for the transfer of the terminal phosphate from a triphosphate nucleotide (eg, ATP) to a diphosphate nucleotide (eg, UDP) via a 'ping-pong' mechanism.28, 29
Among all the NDPK family members from different organisms, the S120 residue is highly conserved. The NDPK-AS120G recombinant protein displays ~50% lower phosphotransferase activity than the wild-type recombinant protein in vitro.23 The same mutation when introduced to NDPK of Dictyostelium discoideum results in an 80% loss of the activity.30 The reduction of NDPK-AS120G activity is caused by the instability of its phosphorylated intermediate, as there are no defects in the phosphate incorporation of the H118 residue nor in the phosphate transfer from NDPK-AS120G to UDP.23 In human neuroblastoma tissues, a decrease in the phosphotransferase activity of NDPK-AS120G apparently is compensated for by other NDPK family members, such as nm23-H2-encoded NDPK-B.23, 31 Therefore, function(s) other than the phosphotransferase activity of NDPK-AS120G likely account for its metastasis-promoting role in neuroblastoma.
All known eukaryotic NDPKs, including NDPK-A, exist in a hexameric quaternary structure via assembling identical dimers.2, 32 However, NDPK-AS120G affects the subunit assembly and results in 17% dimeric structures, which is approximately sixfold higher than the wild type, but only when disulfide bonds are reduced.23 This indicates the susceptibility of the NDPK-AS120G structure to the intracellular redox state. Moreover, NDPK-AS120G reduces its enzyme stability when subjected to heat and urea,23, 33 and exhibits a protein-folding defect.33 This folding defect can be corrected when NDPK-AS120G is phosphorylated by ATP or by phosphoramidate.34 When forming a complex with ADP, no significant structural changes are observed between NDPK-AS120G and the wild-type.35
In addition to affecting the subunit assembly, the S120G mutation changes the interaction of NDPK-A with other cellular proteins. NDPK-AS120G, in contrast to the wild type, interacts with the 28-kDa protein23 but not with PRUNE.36 NDPK-AS120G also appears to indirectly alter protein-protein interaction. For example, the S120G mutation abolishes the ability of NDPK-A to suppress desensitization of the muscarinic potassium current.37 Extracellular recombinant NDPK-AS120G is more efficient than the wild type in supporting the colony formation of undifferentiated human embryonic stem cells.38
NDPK-A-RELEVANT MOLECULAR DETERMINANTS OF NEUROBLASTOMA METASTASIS
For NDPK-A to be a metastasis promoter in neuroblastoma, certain interacting proteins of NDPK-A39 may be the molecular determinants of neuroblastoma metastasis. Data from genome- and exome-wide sequencing studies are useful for identifying these molecular determinants. It has been reported that recurrent mutations affect pathways such as focal adhesions, Rac/Rho, RAS-MAPK, and YAP in advanced and relapsed neuroblastomas.3, 40, 41, 42 Among these pathways, the Rac/Rho pathway is pertinent to the current knowledge of the NDPK-A network.
Rac, Rho, and Cdc42 are well-studied members of the Rho GTPase family, which is a part of the Ras superfamily.43 Rho GTPases regulate cytoskeletal rearrangement, essential for cell migration, invasion, and neuritogenesis,43 and are relevant to neuroblastoma metastasis.
High-frequency recurrent mutations of Tiam1 have been detected in one but not a second study of advanced neuroblastomas due to a low mutation frequency.3, 44 As an interacting protein of NDPK-A,45 Tiam1 functions as a Rac1-specific guanine nucleotide exchange factor46, 47 and participates in neuritogenesis, cell invasiveness, and tumor progression.48 In addition to several genes involved in neuronal growth cone stabilization, Tiam1 and other regulators of the Rac/Rho pathway are also mutated, implicating defects in neuritogenesis.3
Data of chromosomal aberrations in advanced neuroblastoma are also useful for identifying molecules and pathways in the NDPK-A network. The loss of heterozygosity of 1p36 occurs in 23–35% high-risk neuroblastomas.6 One of the genes is located on 1p36 is Cdc42, and its gene product interacts with NDPK-A.49 In MYCN non-amplified neuroblastomas, overexpressed NDPK-A binds to Cdc42 and prevents the induction of neuronal differentiation.50 In advanced neuroblastomas with MYCN amplification, MYCN inhibits neuritogenesis by downregulating Cdc42 expression.50
METASTASIS-RELEVANT NEURAL CREST DEVELOPMENT
Neuroblastoma originates from multipotent neural crest cells committed to the lineage of sympathetic neurons. At the end of the first trimester in humans, neural crest cells are induced to undergo an epithelial-to-mesenchymal transition (EMT), delaminate from the neural tube and migrate through surrounding tissues before arriving at their final destination for terminal differentiation.51 Neural crest development thus shares common mechanisms with tumor metastasis, including EMT, migration, and invasion.52
NDPK-A is highly expressed in the first-trimester placenta in humans, whereas it is downregulated in second- and third-trimester placentas.53 The high level of NDPK-A seen in advanced neuroblastoma indicates deregulation of nm23-H1 expression. If nm23-H1 deregulation occurs during embryogenesis, it will arrest neural crest cells in less differentiated, yet highly migratory and invasive stages, leading to more aggressive neuroblastoma. Ectopically expressed NDPK-A or NDPK-AS120G inhibits neuronal differentiation of NB69 cells upon induction with retinoic acid.22 Such an arrest of the neural crest may occur indirectly via the ability of NDPK-A to bind the c-myc promoter and reduce its transcription (unpublished data), considering that c-Myc is required for neural crest specification.54, 55 Alternatively, NDPK-A-mediated reduction of c-myc transcription possibly increases neuroblastoma metastasis because c-Myc suppresses the metastasis of human breast carcinoma.56
Understanding the developmental functions of NDPK orthologs in different organisms (see review by Ćetković et al in this issue) will clarify the underlying mechanisms of tumor metastasis.
A prognostic signature, consisting of nm23-H1 and two other genes, has been proposed for risk stratification of neuroblastoma patients. NDPK-A is encoded by nm23-H1 and acts as a metastasis promoter in neuroblastoma (eg, an embryonic tumor), unlike its metastasis-suppressing role found in many adult tumors such as breast and prostate carcinoma. Overexpression and the S120G mutation of NDPK-A, likely driving forces of neuroblastoma metastasis (Figure 1), occur in patients with advanced neuroblastoma. Relative to the wild type, NDPK-AS120G is more effective in promoting cell invasiveness and metastasis of neuroblastoma in vitro and in vivo. NDPK-AS120G appears to be a gain-of-function mutation because it increases, whereas the wild-type suppresses, the invasiveness of breast and prostate carcinoma cells. An apparent gain-of-function of the S120G mutation of NDPK-A is likely caused by a protein-folding defect, which affects its protein-protein interactions.
The molecular mechanism(s) by which NDPK-A promotes neuroblastoma metastasis remains elusive. Future studies will be facilitated by identifying molecules and pathways that are frequently altered in advanced neuroblastoma. Because developmental and metastatic processes share common mechanisms, understanding the functions of NDPK orthologs in neural crest development will shed light on neuroblastoma metastasis.
A promising targeted therapy for neuroblastoma is being developed based on a permeable peptide that disrupts the interaction of NDPK-A and PRUNE (reviewed by Ferrucci et al in this issue). This targeted therapy is, unfortunately, not useful for treating neuroblastoma patients harboring the S120G mutation because there is no interaction between NDPK-AS120G with PRUNE. Frequent disruption of Rho GTPases signaling pathways in advanced neuroblastoma suggests potential therapeutic strategies for preventing neuroblastoma metastasis. Because of NDPK-A’s dichotomous role in tumor metastasis as both a suppressor and a promoter, tumor genome/exome profiles are necessary to identify the molecular drivers of metastasis in the NDPK-A network for developing tumor-specific therapies.
The authors thank Larry P Paris for editing the manuscript. This work was supported by the Hung-Hwa Memorial Fund in Taiwan.