Insights into the biology and prevention of tumor metastasis provided by the Nm23 metastasis suppressor gene

Abstract

Metastatic disease is the major cause of death among cancer patients. A class of genes, named metastasis suppressors, has been described to specifically regulate the metastatic process. The metastasis suppressor genes are downregulated in the metastatic lesion compared to the primary tumor. In this review, we describe the body of research surrounding the first metastasis suppressor identified, Nm23. Nm23 overexpression in aggressive cancer cell lines reduced their metastatic potential in vivo with no significant reduction in primary tumor size. A complex mechanism of anti-metastatic action is unfolding involving several known Nm23 enzymatic activities (nucleotide diphosphate kinase, histidine kinase, and 3′–5′ exonuclease), protein–protein interactions, and downstream gene regulation properties. Translational approaches involving Nm23 have progressed to the clinic. The upregulation of Nm23 expression by medroxyprogesterone acetate has been tested in a phase II trial. Other approaches with significant preclinical success include gene therapy using traditional or nanoparticle delivery, and cell permeable Nm23 protein. Recently, based on the inverse correlation of Nm23 and LPA1 expression, a LPA1 inhibitor has been shown to both inhibit metastasis and induce metastatic dormancy.

1. Background

Metastasis is a critical factor in the prognosis of cancer patients and is a major contributor to mortality. Although surgery, radiotherapy, and chemotherapy have improved, the goal of dramatically increasing patient survival remains to be achieved. As such, the metastasis research field has focused on identifying new therapeutic strategies. The discovery of a new class of genes, the metastasis suppressor genes, has therefore received much attention. Nm23 (NME) was the first metastasis suppressor identified [1] on the basis of its reduced expression in a comparative hybridization screen of poorly versus highly metastatic sublines of the murine melanoma cell line K-1735. Transfection and knockout mouse experiments, discussed below, confirmed a metastasis suppressive function. This gene was independently identified as a regulator of imaginal disc differentiation late in Drosophila development as Awd [2, 3]. Thus far, ten human homologues (Nm23-H1–Nm23-H10) have been described with Nm23-H1 and Nm23-H2 being the most studied. They have been separated into two groups based on sequence homology. Group I (H1–H4) includes proteins with nucleotide diphosphate kinase (NDPK) activity and 58–88 % similarity; however, group II (H6–H10) consists of more divergent proteins with only 25–45 % similarity [4].

Since the establishment of Nm23 as the first metastasis suppressor gene, many experiments have been performed attempting to elucidate the exact biochemical mechanism of its anti-metastatic function. Herein, we review the role of Nm23 in the control of metastasis across several cancer cell lines and tumor types. In addition, we examine new therapeutic strategies proposed and tested in in vivo models in order to restore the anti-metastatic function of Nm23-H1.

2. Biochemical functions of Nm23

Multiple biochemical functions have been identified for Nm23 proteins, and several may contribute to the anti-metastatic properties (for a critical review, see [5]). In the 1950s, Drs. Paul Berg and Hans Krebs independently identified a biochemical activity that removed the terminal phosphate from a nucleotide triphosphate (NTP) and added it to a nucleotide diphosphate (NDP). This NDPK activity [6, 7] was subsequently reported to be encoded by Nm23 [8]. Reported functions for the NDPK activity of Nm23 may include maintenance of nucleotide pools, activation of the Gβγ subunit of G proteins by Nm23-H2 [9], and mutator phenotypes [10]. Nm23-H1 is known to be a multifunctional enzyme with at least two other biochemical activities described to date: a histidine protein kinase (HPK) and a 3′–5′ exonuclease. The histidine protein kinases are well known in prokaryotic and lower eukaryotic systems, where they control cellular responses to the environment, but are poorly understood in mammalian cells. The NDPK activity forms the first part of an HPK reaction, in which a high-energy phosphate is transferred from an NTP to Nm23 on a histidine residue. Nm23-P then transfers this phosphate to a protein, either to a high-energy histidine or aspartic acid residue, or possibly an irreversible lower-energy serine or other amino acid phosphorylation. Reported substrates for Nm23 as an HPK include aldolase C, ATP citrate lyase, and the kinase suppressor of Ras [11], the last of which has been linked to Erk-Map kinase activation (review in [12, 13]).

The laboratory of David Kaetzel reported a 3′–5′ DNA exonuclease activity for NDPK-A/Nm23-H1 [14]. Using a series of Nm23-H1 mutations transfected into 1205LU melanoma cells, both the histidine involved in its NDPK and HPK activities and a lysine involved in its exonuclease activity were shown to be partially responsible for metastasis suppression [15]. From these observations, the authors demonstrated, in the melanoma cell line WM793 and in MEFs, a role for the Nm23-H1 NDPK and 3′–5′ exonuclease activities in the repair of UV irradiation-induced DNA lesions [16]. The authors speculate that Nm23-H1 expression reduces the acquisition of mutations that promote transition to the metastatic phenotype in melanoma cells.

The anti-metastatic property of Nm23 has also been associated with its ability to form specific protein–protein interactions (Table 1). In general, ruling out nonspecific interactions due to Nm23 “stickiness” has been critical; bonafide interactions are typically confirmed by two way co-immunoprecipitations and alter the function of one of the proteins involved. A number of pathways potentially involved in metastasis are affected by Nm23 interaction. For several proteins, Nm23-H1 interaction essentially “titers out” the amount of free, prometastatic protein. Examples of such titering include Prune [17, 18] and some viral proteins [ 19, 20]. In other cases, more complex interactions occur modulating cytoskeletal architecture or scaffolds for the Erk-Map kinase pathway.

Table 1.

Nm23-H1 binding proteins and the pathway(s) regulated by the interaction

Protein	Regulated pathway	Phenotype	Isoform	Reference
Tiam1	Rac pathway	Rac1 inactivation	Nm23-H1	[68]
Ksr	Erk pathway	Ksr degradation	Nm23-H1	[11]
Rps3	Erk pathway	Ark pathway inactivation	Nm23-H1	[69]
Lbc	Rho GTPase pathway	Rho GTPase pathway inhibition	Nm23-H2	[70]
Rad	Ras pathway	Ras pathway inactivation	Nm23-H1	[71]
Dbl-1	cdc42	Cdc42 GTPase inhibition	Nm23-H1	[72]
ARF6	Endocytosis	E-cadherin-mediated edocytosis	Nm23-H1	[73]
STRAP	TGFβ pathway	p53-induced apoaptosis and cell cycle arrest	Nm23-H1	[74]
EBNA-1-3C	Transcriptional regulation	Increased MMP9 and αV integrin expression	Nm23-H1	[20]
ERα	Transcriptional regulation	Reduced cathepsin and Bcl-2 expression	Nm23-H1	[75]
ERβ	Transcriptional regulation	Increased estrogen-mediated transcription	Nm23-H2	[76]
RORaa	Transcriptional regulation	N/A	Nm23-H1, Nm23-H2	[77]
Oct-1	Transcriptional regulation	N/A	Nm23-H1	[78]
SET	Transcriptional regulation	Granzyme A-mediated cell death	Nm23-H1	[79]
Vimentin	Cytoskeleton modulation	Increased intermediate filaments density	Nm23-H1	[80]
Tubulin	Cytoskeleton modulation	N/A	Nm23-H1	[81]
Icap-1a	Cell adhesion	N/A	Nm23-H2	[82]
Plakoglobin	Cell adhesion	Increased protein stability	Nm23-H1, Nm23-H2	[83]
APE1	DNA repair	Increased APE1 endonuclease activity	Nm23-H1	[15]
Prune	Pro-metastasis	Increase cAMP-PDE prune activity	Nm23-H1	[84]
MDM2	p53 degradation	Inhibition Nm23 anti-motility ability	Nm23-H2	[85]
HPV 16E7	Cervical cancer	Ubiquitin-proteosome-dependent Nm23 degradation	Nm23-H1, Nm23-H2	[86]
GAPDH	Glycolytic pathway	Phosphoglycerate mutase activation	Nm23-H1	[87]
Aurora-A	Mitosis	N/A	Nm23-H1	[88]

^aThe interaction has been shown only by two-hybrid and GST pull down experiments

A final activity of Nm23 is the regulation of gene expression, which may be influenced by Nm23 participation in transcriptional complexes. The role of Nm23 protein binding as part of a transcriptional complex is only partially understood but may be critical to downstream changes in gene expression. A gene expression profile of MDA-MB-435 cells over-expressing Nm23-H1 or its mutant forms lacking the metastasis suppressive property revealed an inverse correlation between the expression of Nm23-H1 and EDG2 (LPAR1), the first in the family of lysophosphatidic acid receptors [21]. EDG2 over-expression in MDA-MB-435 cells was able to enhance their motility and metastasis [22], suggesting it may serve as a target of Nm23-H1-mediated suppression. Interestingly, recent results showed that the LPAR1 inhibitor Debio 0719 dramatically decreased metastasis in mice [23].

3. Nm23 anti-metastatic properties

3.1. Transfection studies

According to the definition, metastasis suppressors only block the spread of cancer to distant sites without reducing primary tumor formation or size. As such, in vitro experiments (such as soft agar cloning, wound scratch assays, and Boyden chamber chemotaxis assays) may reveal information about component parts of the metastatic process but are not sufficient for studying metastasis. The metastatic cascade is a complex, multi-step process that requires the interaction between cancer cells and the tissue microenvironment. Therefore, in vivo studies are necessary to evaluate the ability of a gene, or chemotherapeutic, to suppress metastasis. Both syngeneic and xenograft animal models have been utilized to assess the in vivo effect of Nm23-H1 in spontaneous (primary tumor formation induced metastasis) and experimental (after injection into the bloodstream) metastasis assays. From these experiments, results showed that cancer cells exogenously expressing Nm23-H1 were significantly inhibited in their capacity to form metastatic colonies, though primary tumor formation was largely unaffected (Table 2). Cell lines from multiple histological types of cancer were metastasis suppressed by Nm23. However, in no experiment did overexpression of Nm23 completely abrogate metastasis. This may indicate the importance of obtaining a critical level of Nm23 over-expression within the cancer cells in order to achieve successful metastatic suppression. More likely, the difference in suppression among cancer types, and within subtypes, can be attributed to the varying pathways active among the differing cancers. Complete inhibition may require cooperation from multiple metastasis suppressor genes acting on several pathways.

Table 2.

Ectopic expression of Nm23 in higH1y metastatic cancer cells reduced metastasis formation

Tumor type	Origin	Cell line	Reduction in metastases (%)	Gene	Reference
Breast carcinoma	Human	MDA-MB-435	50–90	Nm23-H1	[89]
Breast carcinoma	Human	MDA-MB-435	40–100	Nm23-H1, Nm23-H2	[90]
Breast carcinoma	Human	MDA-MB-231- BAG	40–50	Nm23-H1, Nm23-H2	[91]
Breast carcinoma	Rat	MTC/MTLn3	30–70	Nm23-H1, Nm23-H2	[92]
Breast carcinoma	Murine	4T1	60–70	Nm23-H1, Nm23-M1	[23]
Melanoma	Murine	K-1735 TK	58–96	Nm23-H1	[93]
Melanoma	Murine	B16-sublines	54–90	Nm23-M1, Nm23-M2	[94]
Melanoma	Murine	B16F10	93	Nm23-H1	[95]
Melanoma	Human	1205LU	62	Nm23-H1	[15]
Melanoma	Murine	MelJuSo	40–80	Nm23-H1	[96]
Colon	Murine	colon26	55	Nm23-H2	[97]
Colon	Human	HT-29	90	Nm23-H1	[98]
Oral squamous cell carcinoma	Human	LMF4	80–90	Nm23-H2	[25]

Several exceptions to these observations are noteworthy. First, an exception to the consensus of primary tumor data has been reported, where Nm23-H1/Nm23-H2^−/+ knockout mice developed fewer melanomas after exposure to UV radiation [16]. Second, it remains unclear that Nm23-H2 is as potent of a metastasis suppressor as Nm23-H1. Some reports clearly show a metastasis suppressive activity while others do not, and the data are limited by reporting only certain endpoints without full metastasis assays. Lee et al. showed that when stably overexpressing Nm23-H2 in NIH3T3 fibroblasts, HLK3 hepatocytes and oral carcinoma cells (LMF4), a pro-oncogenic effect was observed, namely, tumor formation in nude mice and increased c-myc, cyclin D1, and NF-kB expression [24, 25]. Nm23-H2 silencing did not show the same effect on invasiveness of liver and colon cancer cells indicating a specific role of the H1 isoform as a negative regulator of cancer cell invasion [26]. Finally, in neuroblastoma and hematopoietic tumors, Nm23 overexpression may have the opposite effect.

It is interesting to note that Nm23-H1 has been shown to suppress multiple steps of the metastatic cascade, including invasion, tumor cell survival, and colonization. Multiple transfection studies have described less invasive primary tumors when Nm23 is overexpressed. Horak et al. showed that Nm23 was able to suppress metastasis at the initial steps in metastatic colonization [21]. In experimental metastasis assays, fluorescently labeled MDA-MB-435 cells over-expressing Nm23-H1 showed equivalent arrival in the lungs as control transfectants, but a 10-fold reduction in the retention of tumor cells within murine lungs over time.

Pathway analyses of Nm23 overexpressing tumor cells have suggested multiple downstream effects. Using in vitro studies, Boissan et al. described the role of Nm23 in the maintenance of E-cadherin-mediated intercellular adhesion and limitation of the invasive potential, and identified MAPK, Rac1-GTPase, and Akt signaling as pathways involved in this process [26].

3.2. Nm23-M1 null/SV40 transgenic mouse

The Nm23-H1 knockout mouse model confirmed a role for Nm23-H1 in metastasis suppression. The Nm23-H1 knockout shows a normal phenotype except for defects in mammary gland development [27]. However, a phenotype appeared under two experimental situations promoting hepatocellular carcinoma, either when bred with mice expressing the SV40 virus large T antigen in the liver or when mice are injected with the toxin diethylnitrosamine. In both cases, the development of liver tumors was unaffected, but a two-fold increase in lung metastases was observed [28].

Distinct results were obtained for Nm23-H2. The NME1−/−, NME2−/− double KO showed a perinatal death, while heterozygotes displayed an erythrocyte development phenotype [29]. Analyzing the MEF cells from the double knockout, a reduction in receptor coupled G proteins (beta adrenergic receptor coupled Gs protein) in the plasma membrane was observed and this phenotype was rescued by Nm23-H2 but not Nm23-H1 re-expression [30, 31]. Moreover, the Nm23-H2−/− mouse showed no evident phenotype except for a defect in cytokine production in T cells due to a dysfunction in the KCa3.1 channels [32], although metastasis has not been analyzed.

In conclusion, both transfection and Nm23-H1 knockout mice confirm a metastasis suppressive role for Nm23-H1. Additional supportive data emanate from cell permeable Nm23 proteins, discussed below. The role of Nm23-H2 overlaps but may also be more complex.

3.3. Nm23 expression in tumor and metastatic tissues

Nm23 protein levels have been estimated in numerous tumor cohorts (Table 3). Reductions in Nm23-H1 expression have been significantly associated with aggressive behavior in gastric carcinoma, ovarian cancer, melanoma, breast cancer, and some other cancers [33–35]. Nm23 expression was inversely correlated to tumor aggressiveness using several tumor and patient parameters such as overall survival, disease-free survival, lymph node status, tumor stage, and expression of specific markers (i.e., hormone receptors). Discordant conclusions have been published in cohort studies. The size of cohort used to evaluate the correlation Nm23-metastatic disease can affect the results of the analyses. Antibody specificity, grading schemes, and other technical factors also contribute to varying results. Other studies have examined Nm23 expression in metastatic lesions. Reduced expression of Nm23 in metastases, as compared to histologically similar primary tumors, was observed in gastric, head and neck, non-small cell lung, uterine, and breast carcinomas, and melanoma [36–42].

Table 3.

Nm23 expression in human carcinoma cohort studies

Tumor type	Cohort size	Reference
Inverse correlation between Nm23 expression and metastasis
Breast cancer	27	[99]
Breast cancer	71	[100]
Breast cancer	168	[101]
Breast cancer	390	[102]
Breast cancer	163	[103]
Breast cancer	130	[104]
Breast cancer	128	[105]
Breast cancer	73	[106]
Melanoma	30	[107]
Melanoma	157	[108]
Melanoma	21	[109]
Melanoma	32	[110]
Melanoma	120	[111]
Prostate cancer	346	[112]
Ovarian carcinoma	50	[113]
Gastric cancer	24	[114]
Gastric cancer	59	[36]
Gastric cancer	413	[34]
Hepatocellular carcinoma	21	[115]
Hepatocellular carcinoma	47	[116]
Hepatocellular carcinoma	43	[117]
Non-small-cell lung cancer	381	[118]
Non-small-cell lung cancer	104	[119]
Non-small-cell lung cancer	285	[120]
Oral squamous cell carcinoma	86	[121]
Positive correlation between Nm23 expression and metastasis
Neuroblastoma	76	[43]
Osteosarcoma	25	[44]
Non-small-cell lung carcinoma	36	[122]
Myelogenous leukemia	110	[123]
Peripheral T-cell lymphoma	137	[124]
Peripheral T-cell lymphoma	102	[125]
Lymphomas	606	[126]
Diffuse large B-cell lymphoma	172	[126]
Hodgkin lymphoma	128	[127]
Acute myelogenous leukemia	102	[49]
Thyroid carcinomas	86	[128]

In stark contrast to the above results, high Nm23-H1 levels were associated with a significant reduction in survival for patients with neuroblastoma, osteosarcoma, and hematological malignancies [43–45]. Obviously, Nm23 may play a tissue-specific role, and different regulatory mechanisms may act in different tumors.

Although the amino acid sequence of Nm23 does not contain a conserved secretion signal peptide, it has been detected in conditioned medium of some tumor cell lines and patients’ sera [46–48]. It is unclear how Nm23 protein is secreted into the extracellular space. However, several studies using lymphomas and leukemias evaluated the serum level of Nm23-H1 protein using an ELISA detection system and correlated Nm23 levels with the clinical outcome of patients. One hypothesis is that the major portion of serum Nm23 levels results from erythrocyte death. In vitro experiments using these cancer types suggested that extracellular Nm23 protein may promote lymphoma and leukemia growth and survival by autocrine and/or paracrine mechanisms [49–51]. It has been suggested that extracellular Nm23-H1 indirectly increases the survival of the immature CD34+/CD11b− cells or primary cultured mononuclear cells (PBMNCs), by inducing the production of inflammatory cytokines (IL-1b, IL-6, IL-8, GM-SCF, and MCP-1) from the more mature CD34−/CD11b+ cells.

4. Therapeutic strategies using Nm23-H1

The development of new therapeutic strategies to prevent, halt, or shrink metastases is greatly needed. For the last two decades, several therapeutic strategies using Nm23-H1 have been reported in solid tumor model systems.

4.1. Re-expression of Nm23-H1

The re-expression of Nm23-H1 in breast cancer cells by medroxyprogesterone acetate (MPA) has been reported [52] through the glucocorticoid receptor in triple-negative (estrogen and progesterone receptor negative, HER2 normal) breast cancer. MPA bound to the glucocorticoid receptor and the glucocorticoid response element, altering nucleosomal structure and permitting transcription factor binding to the transcription factor binding sites. Treatment with MPA elevated Nm23-H1 expression in progesterone receptor negative, glucocorticoid receptor positive MDA-MB-231 and MDA-MB-435 human breast carcinoma cell lines in vitro. No effect on proliferation was observed in vitro, but soft agar colonization of metastatic breast cancer cell lines was reduced by approximately 50 % in vitro. MPA had no effect on colony formation of MDA-MB-231T cells expressing antisense Nm23-H1 [53], suggesting some degree of specificity.

Whether MPA-mediated re-expression of Nm-23-H1 would inhibit metastatic progression in vivo was examined through xenograft experiments with MDA-MB-231T cells, in which these cells were intravenously injected into the mice. MPA treatment induced Nm23-H1 expression in MDA-MB-231T cells in vivo, decreasing the metastatic colonization in the lungs of MPA-treated mice by 27–36 % in two independent experiments. Moreover, high levels of Nm23-H1 were detected in 43 % of pulmonary lesions of MPA-treated mice compared with 13 % of metastases in untreated mice. Side effects of MPA treatment included weight gain and decreased bone density. Based on these data, a phase II clinical trial of MPA with metronomic chemotherapy was conducted (personal communication, Dr. Kathy Miller, Indiana University).

Multiple other compounds and drugs have been reported to elevate Nm23 expression. These include thujone, a monoterpene natural product. Injection of thujone into mice bearing B16F-10 melanoma cells improved survival and upregulated Nm23 among other molecular changes [54]. Another natural product, the polysaccharide LMPAB, inhibited B16 melanoma metastasis, elevated Nm23 expression, and inhibited protease expression [55]. Again, using the B16 model system, amentoflavone significantly lowered pulmonary metastases and increased Nm23 expression, among other markers [56]. In vitro data also identified the anti-inflammatory compounds indomethacin and acetylsalicylic acid, a vitamin D analog, all-trans retinoic acid, the anti-oxidant l-carnosine, and gamma linolenic acid as up-regulating Nm23 proteins. All of these compounds are nonspecific, in that multiple additional mechanisms of action are anticipated, and the sum of risks and benefits will determine efficacy rather than effects on a single pathway.

4.2. Gene therapy

Two Nm23 delivery methods have been reported. One is an adeno-associated virus (AAV)-mediated transduction and the other is a non-viral nanoparticle-mediated transduction. The effectiveness of the former method was examined through an orthotopic transplantation model with SW626-M4 human ovarian adenocarcinoma cell lines expressing low Nm23, in which the tumor fragments formed by these cells in nude mice were implanted in the ovarian serosa. In more than 95 % of ovarian tumor cells in mice intraperitoneally injected with AAV-Nm23-H1, increased Nm23 expression was observed. The experiments demonstrated that the number of animals developing liver metastasis in the AAV-Nm23-H1-treated group reduced to 60 % of that in the PBS- or AAV-LacZ-treated group, and that the AAV-Nm23-H1 treatment increased the time and percentage of survival (46 %, at 140 days) as compared with the PBS- or AAV-LacZ-treated animals (0 %, all died 35 days earlier) [57].

The effectiveness of the nanoparticle method was examined through experimental metastasis experiments with the B16F10 mouse melanoma cell line. The nanoparticle was a poly-l-lysine-modified iron oxide nanoparticle (IONP-ALL), which was formed by modifying poly-l-lysine to the surface of iron oxide nanoparticles. Positively charged IONP-PLL bound to negatively charged DNA phosphate groups and showed greater resistance to DNase-1-mediated DNA degradation compared with naked DNA. IONP-PLL/DNA complexes that were intravenously injected into mice were distributed into multiple organs such as the brain, lungs, spleen, and kidneys [58]. In metastasized B16F10 cells in the lung derived from the mice intravenously injected with IONP-PLL carrying Nm23-H1 plasmid, strong expression of Nm23 was observed. In the IONP-PLL/NM23-H1-treated group, metastasis was clearly suppressed compared with the group treated with free Nm23-H1 plasmid. The groups of mice treated with empty IONP-PLL, naked Nm23 plasmid DNA, or saline all developed metastases similarly. The intravenous injection of IONP-PLL carrying Nm23-H1 plasmid significantly extended the survival time of the mice inoculated with B16F10 [59]. Although the delivery system also leads to Nm23 expression in normal tissues, no side effects have been observed. Both approaches are limited by general problems with gene therapy strategies.

4.3. Protein therapy using cell permeable Nm23

The effectiveness of protein-based therapy to deliver cell permeable Nm23 protein (CP-Nm23) as an anti-metastatic agent has been reported [60]. CP-Nm23 is the fused protein composed of hydrophobic macromolecule transduction domains (MTD) and Nm23. MTD is an improved amino acid sequence derived from a membrane-translocating motif composed of 12 amino acids from a hydrophobic signal sequence from fibroblast growth factor 4. The MTD fused with eGFP was quickly incorporated into several human cancer cell lines such as MDA-MB-435 (breast cancer cells), HCT116 (colon cancer cells), and A549 (lung cancer cells) in vitro, and murine organs such as the liver, spleen, and lung in vivo [60].

Biological effects of CP-Nm23 were examined through in vitro experiments using MDA-MB-231 and MDA-MB-435 human breast cancer cell lines and xenograft experiments with MDA-MB-231 cells. Both cell lines incorporating CP-Nm23 proteins reduced MEK and ERK phosphorylation, consistent with Ksr data, and EDG2 and gene expression alterations previously described. CP-Nm23 inhibited metastasis-associated phenotypes including cell migration, adhesion, matrigel invasion, and anchorage-independent growth in vitro. Moreover, CP-Nm23 not only suppressed the establishment of lung metastases in the in vivo models but also cleared already established metastases, thereby greatly prolonging the survival of animals harboring disseminated tumor cells. Indeed, the mice injected with CP-Nm23 showed enhanced survival (90 %) after 40 weeks as compared with control mice (20 %). Bouin’s solution and vimentin staining confirmed the reduction or lack of lung metastases in the treated mice. [60]

4.4. LPA1, inversely expressed with Nm23, as a therapeutic target

An inverse correlation between Nm23-H1 levels and the expression of a therapeutic target in inhibiting metastasis progression was reported [22]. The target is known as LPA1 (also known as EGD2 or LPAR1), one of the G protein-coupled receptors for serum lysophosphatidic acid (LPA). LPA is produced from lysophosphatidylcholine in the cell membrane and in plasma by autotaxin. It is now known that the effects of LPA at physiological concentrations are mediated by five bona fide, high-affinity cognate receptors (LPA1–LPA5), low-affinity cognate receptors (P2Y5, P2Y10, and GPR87) and perhaps by additional recently proposed or as yet unidentified receptors [61, 62]. These signaling pathways have been shown to prompt aspects of tumorigenesis and metastatic progression [63].

Gene expression profiling of MDA-MB-435 cells expressing wild-type Nm23-H1, compared with those expressing mutant forms of Nm23-H1 (P96S and S120G) lacking the anti-motility property, identified LPA1 as downregulated by wild-type but not by mutant Nm23-H1 expression. In vitro experiments demonstrated that LPA1 overexpression significantly restored motility to MDA-MB-435 cells expressing Nm23-H1. Further, LPA1 knockdown via RNA interference reduced the motility of MDA-MB-435 and MDA-MB-231 cells. Moreover, in serial sections of human breast cancer specimens, an inverse pattern of Nm23-H1 and LPA1 staining was observed. In hepatocellular carcinomas developed from Nm23 knockout mice, elevated expression of LPA1 was also observed. Given the above data, the hypothesis that an LPA1 inhibitor can prevent the metastatic progression of breast cancer cells was examined through spontaneous animal experiments with 4T1 murine mammary carcinoma and xenograft experiments with MDA-MB-231T cells [23].

Debio 0719 (Debiopharm S.A., Lausanne, Switzerland) is an antagonist for LPA receptors and selectively inhibits LPA receptor-mediated actions, especially through LPA1 and LPA3. The IC₅₀ of 0719 for LPA1 is 60 nM, with less potent inhibition of LPA3 at 660 nM and LPA2 at 2 μM. In vitro experiments demonstrated that 0719 had no effect on proliferation of 4T1 and MDA-MB-231T cells but inhibited their motility. In a spontaneous metastasis assay using 4T1 murine mammary cells, 0719 had no effect on primary tumor formation but inhibited lung metastasis by 89 % and liver metastasis by 73 %. The metastasis inhibitory activity of 0719 was confirmed in MDA-MB-231T experimental metastasis assays using both lung metastasis counts and animal survival as endpoints.

Analysis of the histologic section of the primary and metastatic tumors derived from the mice inoculated with 4T1 cells revealed that the Ki67 labeling of primary tumors from mice treated with vehicle or 0719 was comparable (P=0.86). In contrast, in vehicle-treated liver metastases, 15.3 % of tumor cells were Ki67 positive and declined 65 to 5.4 % in the 0719-treated lesions (P=0.005). Similar trends were observed in 4T1 lung metastases. No detectable cleaved caspase 3 was observed at any site. Thus, LPA1 pathway interruption halted tumor proliferation in a site-specific manner, suggestive of an induction of metastatic dormancy. Dormancy has been proposed to result from the reciprocal down-regulation of the proliferative Erk-Map kinase pathway and up-regulation of the p38 stress Map kinase pathway [64–67]. This signaling pattern was observed in 4T1 cells in the liver and lung of 0719-treated mice but not primary tumors. The dormancy status induced by 0719 treatment was shown to be reversible, and discontinuation of 0719 administration in experimental metastasis assays with MDA-MB-231T cells led to a decrease in survival with pulmonary metastasis evident at necropsy [23].

5. Conclusion

The metastasis suppressor field has identified a series of proterns with non-obvious mechanisms of action that are teaching us new lessons about the regulation of metastatic colonization and providing new therapeutic leads. For solid tumors, Nm23-H1 is unarguably a metastasis suppressor with a complex mechanism of action. New translational approaches that have been developed around this gene, while still in their infancy, have shed light on new approaches to prevent or halt metastatic progression (Fig. 1).

Fig. 1.

Several therapeutic strategies using Nm23-H1 have been reported and tested in in vivo models in order to restore the metastatic suppression property of Nm23. Nm23-H1 expression increased after the treatment with Medroxyprogesterone acetate (MPA) or other natural compounds ( thujone, LMPAB, amentoflavone). Three Nm23-H1 delivery methods, the adeno-associated virus (AAV)- Nm23-H1, the poly-L-lysine-modified iron oxide nanoparticle (IONP-PLL)-Nm23-H1 and the cell permeable Nm23-H1 protein (CP-Nm23), have been reported. Lysophosphatidylcholine receptor 1 (LPA1), a new therapeutic target mediator of metastatic dormancy was identified , based on its inverse correlation to Nm23 expression

New tools are needed in the metastasis field for continued progress. These include tools for the analysis of phosphohistidine and the HPK function of Nm23. More generally, newer metastatic models that recapitulate important aspects and the heterogeneity of human disease will help prove the generality of translational findings.