ARTICLE

Increased Lung Metastasis in Transgenic NM23-Null/SV40 Mice with Hepatocellular Carcinoma

Mathieu Boissan, Dominique Wendum, Sandrine Arnaud-Dabernat, Annie Munier, Marcel Debray, Ioan Lascu, Jean-Yves Daniel, Marie-Lise Lacombe

Affiliations of authors: Unité Institut National de la Santé et de la Recherche Médicale 680, Faculté de Médecine Saint-Antoine, Université Pierre et Marie Curie, Paris, France (MB, AM, M-LL); Laboratoire d'Anatomie Pathologique, Hôpital Saint-Antoine, AP-HP, Paris, France (DW); Laboratoire de Biologie de la Différenciation et du Développement, Université de Bordeaux-2, Bordeaux, France (SA-D, J-YD); IBGC-CNRS, Université de Bordeaux-2, Bordeaux, France (IL); Laboratoire de Biomathématiques, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, France (MD)

Correspondence to: Marie-Lise Lacombe, PhD, INSERM U.680, Faculté de Médecine Saint-Antoine, 27 rue Chaligny, 75571 Paris cedex 12, France (e-mail: lacombe{at}st-antoine.inserm.fr).


    ABSTRACT
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 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background: The metastasis-suppressing role of the NM23 gene in the metastatic spread of solid tumors is still debated. We examined the role of NM23 in tumor development and metastatic dissemination by using transgenic mice that lack mouse NM23 (NM23-M1) in two mouse models of hepatocellular carcinoma (HCC) that recapitulate all steps of tumor progression. Methods: We induced HCC in mice that contained (NM23-M1+/+) or lacked (NM23-M1–/–) NM23-M1 by diethylnitrosamine injection or by a crossing scheme that transferred a transgene that leads to liver expression of simian virus 40 large T antigen (ASV mice). We used microscopic examination and immunohistochemistry to analyze tumor progression. Expression of Nm23 protein isoforms (Nm23-M1 and Nm23-M2) and several tumor markers was analyzed in the primary tumor and in metastases by Western blotting. The statistical significance of differences in the incidence of Nm23-M2 overexpression in null mice relative to that in wild-type mice was tested by a one-sided Fisher's exact test. The statistical significance of differences in the incidence of metastases was examined using one-sided chi-square tests. All other statistical tests were two-sided. Results: In both models, Nm23-M1 and/or Nm23-M2 were overexpressed in the primary liver tumors compared with nontumor liver tissue; however, the lack of the NM23-M1 gene had no effect on primary tumor formation in either model. ASV mice developed pulmonary metastases that were positive for the Hep-Par 1 antibody, which recognizes a specific hepatocyte antigen, whereas the few pulmonary nodules that developed in diethylnitrosamine-injected mice were negative for this antigen. Statistically significantly more ASV/NM23-M1–/– mice than ASV/NM23-M1+/+ mice developed lung metastases (69.2% versus 37.5%; difference = 31.7%, 95% confidence interval = 13.1% to 50.3%; P<.001). In ASV/NM23-M1+/+ mice, immunohistochemical staining for Nm23-M1 was highly heterogeneous among the primary liver tumors, but weak or negative among lung metastases. Conclusions: The lack of NM23-M1 expression promotes metastasis in the SV40 animal model of liver carcinogenesis.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
NM23 was the first gene to be proposed as a metastatic suppressor because of its reduced expression in highly metastatic murine melanoma cell lines (1). In human tissues, the two most abundantly expressed NM23 genes are NM23-H1 (2) and NM23-H2 (3), also known as NME1 and NME2, respectively. These genes encode the A and B subunits, respectively, of nucleoside diphosphate kinase (NDPK) (4,5). The murine orthologs, NM23-M1 (1) and NM23-M2 (6), encode proteins that share 94% and 98% identity, respectively, with their human counterparts. Some studies have reported an inverse association between NM23-H1 expression at the mRNA or protein level and the metastatic potential of human solid tumors such as melanoma and breast, liver, and colon carcinomas [reviewed in (7,8)]. However, other studies on these and other tumor types have failed to show such a relationship. Indeed, neuroblastoma aggressiveness was found to be directly associated with NM23-H1 expression (9,10). Nevertheless, in agreement with studies showing an inverse association between NM23 expression and metastasis, aggressive tumor cell lines that were engineered to overexpress NM23-H1 or NM23-M1 displayed reduced metastatic potential in experimental models of metastasis (1114).

In vitro, NM23 overexpression results in reduced anchorage-independent colonization in response to transforming growth factor-{beta}, reduced motility in response to growth factors, and increased differentiation [reviewed in (8)]. Two findings from several additional studies suggest that NM23 has a dual role in tumor progression: 1) its overexpression in primary tumors at early stages, and 2) the association between the loss of NM23-M1 expression in later stages and tumor aggressiveness and metastatic potential. The involvement of the closely related gene NM23-H2 in metastatic dissemination is less well documented and remains unclear (19).

The mechanisms by which NM23 may influence metastatic potential are largely unknown. Like the enzymes responsible for triphosphate nucleoside synthesis, particularly GTP synthesis, Nm23 isoforms could play a role in proliferation and signal transduction (20,21). Nm23 isoforms are found in complexes with proteins involved in endocytosis (22), in cell migration (particularly with Tiam1, the specific GDP/GTP exchange factor of Rac1) (23), and in cell adhesion (24,25). Nm23-H1 also interacts with kinase suppressor of Ras (KSR), a scaffold protein of the extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway (26). Nm23 isoforms interact with DNA and may regulate gene expression. Nm23-H1 binds to the promoters in the matrix metalloproteinase 2 gene [MMP-2 (27)] and the platelet-derived growth factor A gene [PDGF-A (28,29)]; Nm23-H2 is identical to PuF, a transcription factor that controls transcription of the c-myc gene (30,31).

To further analyze the role of NM23 in primary tumor development and metastatic dissemination, we studied hepatic tumor progression in NM23-M1 null mice with hepatocellular carcinoma (HCC). We used two well-characterized mouse models of hepatocarcinogenesis. In one model, diethylnitrosamine injection was used to induce HCC (32) in mice that contained or lacked NM23-M1; in the other model, mice lacking the NM23-M1 gene were crossed with transgenic mice that express the simian virus 40 (SV40) T antigen in the liver and spontaneously develop HCC (33). In both models, mice develop lung nodules, therefore reproducing all the steps from primary solid tumor formation to metastatic spread.


    MATERIALS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals

Transgenic NM23-M1 null mice (NM23-M1–/– mice; mixed 129Sv-C57BL/6J genetic background) were obtained and characterized as previously described (34). Transgenic ASV mice (C57BL/6J-DBA2 genetic background) harboring the SV40 early region coding for the large T antigen under the control of the human antithrombin III gene regulatory sequences were a generous gift from Dr. P. Briand (Institut Cochin, Paris, France) and have been described in detail elsewhere (33). The SV40 transgene is located on the Y chromosome, and all transgenic male mice systematically develop HCC beginning at 4 months of age. All animals were handled in compliance with national ethical guidelines for the care and use of laboratory animals. Food and water were available ad libitum. Before the mice were killed, they were anesthetized by intraperitoneal injection of a mixture of ketamine (72 mg/kg) and xylazine (4.8 mg/kg). This study was approved by the Bureau de l'Expérimentation Animale of the Institut National de la Santé et de la Recherche Médicale (INSERM).

Chemical Induction of Hepatocarcinogenesis

The carcinogenic and genotoxic agent diethylnitrosamine (Sigma-Aldrich, St. Louis, MO) was diluted in 0.9% NaCl and injected intraperitoneally as a single dose of 10 µg/g body weight into 15-day-old male NM23-M1+/+ and NM23-M1–/– mice as previously described (32). We injected sibling mice to avoid genetic background–specific differences.

Spontaneous Hepatocarcinogenesis in ASV Mice

We generated double transgenic ASV/NM23-M1–/– mice by first crossing female NM23-M1–/– mice with male ASV mice to create male ASV/NM23-M1+/– mice and female NM23-M1+/– mice, which were crossed to each other to generate double transgenic F2 ASV/NM23-M1–/– male mice that were compared with ASV/NM23-M1+/– and ASV/NM23-M1+/+ male mice. Siblings were compared with each other to limit genetic background-dependent variations in tumor formation. The NM23-M1–/–, NM23-M1+/–, and NM23-M1+/+ female siblings, which did not develop HCC, were used as controls. Mouse genotyping was performed on genomic DNA isolated from mouse tails using polymerase chain reaction (PCR) and the following oligonucleotide primer sets: primer set 1, which yielded a 5454-base pair (bp) PCR product corresponding to the wild-type allele (forward primer: 5'-GGCG GTAAAGCCTTGTCAT-3'; reverse primer: 5'-AGCAACCACT GGTCCTGAGT-3') and primer set 2, which yielded a 2600-bp PCR product corresponding to the NM23 deletion-specific neo-containing cassette (34) (forward primer: 5'-CGTTGGCT ACCCGTGATATT-3'; reverse primer: 5'-AGCAACCACTGGT CCTGAGT-3'). The genotype of all homozygous ASV/NM23-M1–/– mice was confirmed by performing two independent PCR analyses.

Histologic and Immunohistochemical Analyses

We removed four randomly selected samples from the liver lobes of each mouse, every month after the third month of age in the diethylnitrosamine-treated mice and from 2-, 3-, 4-, 5-, and 6-month-old ASV mice and subjected them to histopathologic examination. We also removed the five pulmonary lobes in entirety from each mouse for analysis. All tissue samples were fixed immediately in 4% paraformaldehyde for 24 hours and then embedded in paraffin and cut into 4-µm thick sections.

Tissue sections were stained with hematoxylin–phloxine–saffron. Liver tumors were classified according to the International Classification of Rodent Tumors (35). Liver tumors (from diethylnitrosamine-injected mice) and lung nodules (from diethylnitrosamine-injected and ASV mice) were counted. The maximal diameters of ASV lung nodules were assessed by viewing them through an upright light microscope (Axioskop, Zeiss, Jena, Germany) using the 40x objective equipped with an eyepiece that contained a square frame of 0.3 mm per side, allowing us to easily sort small metastases (less than or equal to 0.3 mm) from larger ones (greater than 0.3 mm). Approximately 50 mice of each genotype (ASV/NM23-M1+/+, ASV/NM23-M1+/–, and ASV/NM23-M1–/–) were used to study lung metastasis.

For immunohistochemical analyses, 4-µm thick tissue sections were dewaxed in xylene, rehydrated in a graded alcohol series, and then incubated in 0.1% hydrogen peroxide in methanol for 30 minutes to inhibit endogenous peroxidase activity. We used an Avidin/Biotin Blocking Kit to prevent nonspecific binding (Vector Laboratories, Burlingame, CA). Briefly, sections were incubated for 15 minutes in an avidin D solution, rinsed briefly, and then incubated for an additional 15 minutes in a biotin solution. The following primary antibodies were used: Hep-Par 1 (hepatocyte-paraffin 1, clone OCH1E5, Dako, Glostrup, Denmark), a mouse monoclonal antibody that recognizes a component of hepatocellular mitochondria that is absent in mitochondria of other tissues, used at a dilution of 1:150 and a rabbit polyclonal anti-human Nm23-H1 antibody described in the following section, used at a dilution of 1:3000. Both antibodies cross-react with the respective mouse antigens. Microwave antigen retrieval (750 W for 15 minutes followed by 150 W for 15 minutes in 10 mM citrate buffer, pH 6) was used before sections were incubated with the Hep-Par 1 antibody. Immunolabeling was performed using a Supersensitive Link-Label Immunohistochemistry Detection System (Biogenex, San Ramon, CA) according to the manufacturer's protocol. Briefly, after incubation with the primary antibody, sections were rinsed and then incubated for 20 minutes in a prediluted biotinylated anti-immunoglobulin solution. Sections were rinsed and then incubated for 20 minutes in a prediluted horseradish peroxidase–labeled streptavidin solution. The sections were then incubated in a 3-amino-9-ethylcarbazole solution to reveal peroxidase activity, which was detected as a reddish-brown end product. Sections were counterstained with hematoxylin and mounted with coverslips using an aqueous mounting media (Glycergel, Dako, Glostrup, Denmark). Negative controls included omission of the primary antibody for both Hep-Par 1 and Nm23 labeling and the use of NM23-M1–/– mouse tissue sections for Nm23 labeling.

Western Blotting

Liver samples were rapidly removed from each mouse, immediately frozen in liquid nitrogen, and then lysed in a Dounce homogenizer with cold RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 10 µg/mL leupeptin) and centrifuged at 10 000g at 4 °C for 10 minutes. The protein content of the resulting lysate was determined by the bicinchoninic acid method (Pierce, Biotechnology, Inc., Rockford, IL). Lysates were diluted in Laemmli sample buffer that contained (Nm23-M1 detection) or lacked 8 M urea (detection of all other proteins) and loaded onto 10% SDS–polyacrylamide gels and separated by polyacrylamide gel electrophoresis.

For detection of Nm23-M1 and Nm23-M2, approximately 5µg protein/lane was transferred overnight to PVDF membranes (Millipore Corporation, Bedford, MA) at 4°C and 100 mA in 25 mM potassium phosphate buffer, pH 7.0, and the membranes were fixed for 45 minutes with 0.05% glutaraldehyde in the same buffer. Membranes were then incubated using selective polyclonal antihuman Nm23-H1 and Nm23-H2 antibodies (each used at 1:10 000 dilution) that were raised against human recombinant Nm23-H1 and Nm23-H2 proteins and affinity-purified and depleted of cross-reacting antibodies as previously described (36). These antibodies also specifically recognize the respective mouse antigens, as shown by selective detection of murine recombinant Nm23-M1 and Nm23-M2 proteins used as controls. For the detection of the other proteins (i.e., cyclin D1, cyclin E, cyclin A, Cdk2, p21, c-Myc, Rho-A, Rho-C, phosphorylated ERK1/2, and total ERK1/2), proteins (40 µg protein/lane) were transferred to nitrocellulose membranes at 4 °C and 50 V for 90 minutes, and the membranes were then incubated with the following primary antibodies: mouse monoclonal anti-cyclin D1 (1:100 dilution); rabbit polyclonal anti-cyclin E (1:100 dilution); rabbit polyclonal anti-cyclin A (1:100 dilution); rabbit polyclonal anti-Cdk2 (1:100 dilution); rabbit polyclonal anti-c-Myc (1:100 dilution); mouse monoclonal anti-Rho A (1:100 dilution); goat polyclonal anti-Rho C (1:100 dilution); mouse monoclonal anti-p-ERK1/2 (Tyr204), which recognizes the phosphorylated tyrosine 204 of ERK1/2 (1:500 dilution); rabbit polyclonal anti-ERK1/2 (1:500 dilution) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA); and mouse monoclonal anti-p21 (1:250 dilution; BD Biosciences Pharmingen, San Diego, CA). Primary antibody binding was visualized by enhanced chemiluminescence (Amersham Biosciences, Saclay, France) after incubation of the membranes with a horseradish peroxidase–conjugated anti-rabbit or anti-mouse IgG secondary antibody (Cell Signaling Technology, Inc., Beverly, MA) or horseradish peroxidase–conjugated anti-goat IgG antibody from Santa Cruz Biotechnology, Inc.

NDPK Activity Measurement

NDPK activity in mouse liver lysates was measured using a spectrophotometric coupled pyruvate kinase–lactate dehydrogenase assay as previously described (37) with minor modifications. In brief, the assays were performed in a 1-mL reaction mixture that contained 50 mM HEPES, pH 7.4, 75 mM KCl, 5 mM MgCl2, 1 mM phosphoenolpyruvate, 0.1 mM NADH, 1 mM ATP, 0.2 mM dTDP, 1 mg/mL bovine serum albumin, and 2 units each of pyruvate kinase and lactate dehydrogenase. The reaction was started by adding 10 µL of a liver lysate that was prepared in RIPA buffer as described above. NADH oxidation, which reflects the formation of adenosine diphosphate by NDPK, was followed spectrophotometrically by the decrease in absorbance at 334 nm.

Quantitative PCR Analysis of mRNA Levels

Expression of NM23-M1 and NM23-M2 mRNAs was analyzed by real-time reverse transcription–PCR (RT-PCR). Total RNA was extracted from liver samples with the use of an RNAqueous-4PCR kit (Ambion, Inc., Austin, TX); RNA samples (2 µg) were treated with DNase and then reverse transcribed by extension of random decamers using Moloney murine leukemia virus reverse transcriptase (RETROscript, Ambion, Inc.). We used a LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) with SYBR Green as the fluorophore and the following oligonucleotide primers to amplify the resulting complementary DNAs for NM23-M1 (forward primer: 5'-AGGACCAGTGGTTGCTATGG-3'; reverse primer: 5'-CGCACAGCTCTTGTACTCCA-3') and NM23-M2 (forward primer: 5'-GGCCTCTGAAGAACACCTGA-3'; reverse primer: 5'-GATGGTGCCTGGTTTTGAAT-3'). Each sample was normalized on the basis of its endogenous {beta}-actin mRNA content.

Experimental Metastasis Assay

B16F10 murine melanoma cells (C57BL/6J background; a gift from Dr. M. F. Poupon, Institut Curie, Paris, France) were suspended in serum-free RPMI 1640 medium and injected into the retroorbital sinus of NM23-M1+/+ and NM23-M1–/– mice of pure C57BL/6J genetic backgrounds (105 cells in 0.1 mL/mouse) recently obtained by at least 10 backcrosses. On day 14 after injection, the mice were killed and their lungs were removed and subjected to histologic analysis.

Statistical Analysis

McNemar's exact test (38) was used to test the statistical significance of differences in the incidence of overexpression of Nm23-M1 and Nm23-M2 proteins among NM23-M1+/+ mice. The statistical significance of the higher incidence of Nm23-M2 overexpression in NM23-M1–/– mice relative to that in NM23-M1+/+ mice was tested with a one-sided Fisher's exact test because we were interested in whether or not there was an increased incidence of Nm23-M2 protein overexpression in NM23-M1–/– mice as compared with NM23-M1+/+ mice. NDPK activity was analyzed by using a two-way analysis of variance, followed by multiple pairwise comparisons with Bonferroni adjustment. We performed three one-sided chi-square tests with Bonferroni adjustment to test whether the incidence of lung metastases was statistically significantly higher among ASV/NM23-M1–/– mice than among NM23-M1+/– and NM23-M1+/+ mice and statistically significantly higher among ASV/NM23-M1+/– mice than among ASV/NM23-M1+/+ mice. We used one-sided tests because we were testing whether or not there was an increased incidence of metastases when considering the previously mentioned comparisons. The Mann–Whitney U test was used to identify statistically significant differences in the number of metastases per mouse among ASV/NM23-M1+/+ and ASV/NM23-M1–/– mice. In all tests, the level of statistical significance was set at P<.05. All statistical tests were two-sided except where indicated otherwise.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Primary Tumor Formation in Mice That Contain or Lack the NM23-M1 Gene

We used two murine models of hepatocarcinogenesis to examine the role of the NM23-M1 gene in hepatic tumor formation. In the first model, hepatocarcinogenesis was chemically induced by injection of diethylnitrosamine. We observed no histologic differences in liver tumor development between NM23-M1+/+ and NM23-M1–/– mice. We detected no abnormalities in any mice younger than 6 months. In both groups of mice, basophilic foci of altered hepatocytes were identified in 6-month-old mice, hepatocellular adenomas were identified in 8-month-old mice, and HCC was identified in 12- to 13-month-old mice (data not shown). As shown in Table 1, NM23-M1+/+ mice and NM23-M1–/– mice had similar mean liver weights (expressed as a ratio of liver weight to total body weight) at 3 months of age (3.7% and 3.8%, respectively) and at 13 months of age (10.4% and 11.0%, respectively). The number and area of non-HCC liver nodules (which include basophilic foci and adenomas) and of HCC nodules among 12- to 13-month-old mice were similar for NM23-M1+/+ mice and NM23-M1–/– mice (Table 2).


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Table 1.  Liver weight/total body weight (%) among diethylnitrosamine-treated and ASV mice*

 

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Table 2.  Liver nodule characteristics among diethylnitrosamine-treated mice*

 
In the second model of hepatocarcinogenesis, tumor formation was induced by crossing NM23-M1–/– mice with ASV mice. Among the F2 mice, we observed histologic lesions consisting of hepatocellular atypias in those younger than 2 months, foci of hepatocellular alterations in those 3 months old, and HCC nodules in those 4 months or older, regardless of NM23-M1 genotype (data not shown). HCC nodules invaded the whole liver parenchyma and could not be counted because only tumor tissue was macroscopically visible. We therefore evaluated primary tumor development by measuring the ratio of liver weight to body weight (Table 1). This ratio increased between 2 and 4 months of age, from 7.2 to 30.1 in ASV/NM23-M1+/+ mice and from 9.9 to 25.6 in ASV/NM23-M1–/– mice and then plateaued until 6 months of age in both ASV/NM23-M1+/+ and ASV/NM23-M1–/– mice. There was no difference in this ratio between the two groups during the tumor development time-course. A total of 16 (26%) of 61 ASV/NM23-M1+/+ mice and 17 (26%) of 66 ASV/NM23-M1–/– mice died by the age of 6 months.

Nm23-M1 and Nm23-M2 Protein Expression During Mouse Hepatocarcinogenesis

We used specific polyclonal antibodies raised against recombinant human Nm23-H1 and Nm23-H2 proteins (36) and Western blotting to detect expression of the analogous mouse proteins during primary tumor development in the two mouse models. We included recombinant mouse Nm23-M1 and Nm23-M2 proteins on all gels as controls to verify the specificities of the antibodies and detected no cross-reactivity with either antibody (Fig. 1, A). As expected, we detected no Nm23-M1 protein in liver extracts from NM23-M1–/– mice (data not shown). Among diethylnitrosamine-treated NM23-M1+/+ mice, HCC lysates had higher levels of Nm23-M1 and Nm23-M2 proteins, especially Nm23-M1 protein, than lysates made from nontumor tissue (Fig. 1, A). Among 20 diethylnitrosamine-injected NM23-M1+/+ mice, 18 (90%) expressed higher levels of Nm23-M1 in HCC than in nontumor tissue, whereas only six mice (30%) expressed higher levels of Nm23-M2 in HCC than in nontumor tissue (difference = 60%, 95% confidence interval [CI = 39% to 81%]; P<.001). Quantitation of the data by densitometry scanning showed mean increases of 3.6-fold [95% CI = 3.3-fold to 3.9-fold] and 1.6-fold [95% CI = 1.0-fold to 3.2-fold] in Nm23-M1 and Nm23-M2 protein expression, respectively, in HCC compared with nontumor tissue from NM23-M1+/+ mice. By contrast, eight (67%) of 12 NM23-M1–/– mice injected with diethylnitrosamine expressed higher levels of Nm23-M2 in HCC than in nontumor tissue. Quantitation of the data by densitometry scanning showed a mean 3.3-fold [95% CI = 2.1-fold to 5.9-fold] increase in Nm23-M2 protein expression in HCC compared with that in nontumor tissue from NM23-M1–/– mice. The higher incidence of Nm23-M2 overexpression in NM23-M1–/– mice relative to NM23-M1+/+ mice (P = .034) suggests that there was a compensatory increase in Nm23-M2 protein expression in the tumor as compared to nontumor tissue in the Nm23-M1 deficient mice. We also observed overexpression of the Nm23-M1 and Nm23-M2 genes at the mRNA level (data not shown). During the preneoplastic phase (i.e., in 6- and 8-month-old mice), we observed no increase in the liver levels of Nm23-M1 in NM23-M1+/+ mice and in the liver levels of Nm23-M2 in NM23-M1+/+ and NM23-M1–/– mice compared with that in normal liver from uninjected 6-month-old male mice (Fig. 1, B).



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Fig. 1. Nm23 protein levels in liver extracts from diethylnitrosamine-treated mice. Liver tissue homogenates were prepared from NM23-M1+/+ mice (+/+) and NM23-M1–/– mice (–/–) at different stages of diethylnitrosamine-induced hepatocarcinogenesis and analyzed for Nm23-M1 and Nm23-M2 protein expression by Western blotting with anti-Nm23-H1 and anti-Nm23-H2 antibodies, respectively. A) Nm23 expression in nontumor (N) and hepatocellular carcinoma (T) tissue extracts from 12- or 13-month-old diethylnitrosamine-injected NM23-M1+/+ mice (m1–m4) and NM23-M1–/– mice (m5–m8). Recombinant murine Nm23-M1 (M1) and Nm23-M2 (M2) proteins were used as controls for antibody specificity. B) Nm23 protein expression in livers from 6- and 8-month-old diethylnitrosamine-treated mice and in normal liver from a 6-month-old uninjected male mouse (C). All blots were reprobed with an anti-extracellular signal–regulated kinase (ERK) antibody (which detects both ERK1 and ERK2) to ensure equivalent protein loading.

 
In ASV/NM23-M1+/+ mice, Nm23-M1 protein levels did not change during HCC development as compared with those in normal liver from 3-month-old female mice that do not develop HCC (Fig. 2, A). By contrast, Nm23-M2 levels were very low in female control mice of NM23-M1+/+ and NM23-M1–/– genotypes and increased from 2 months of age until HCC onset (at 5 months of age) in male ASV/NM23-M1+/+ and ASV/NM23-M1–/– mice (Fig. 2, A). Quantitation of the data by densitometry scanning showed 7.5- and 2.7-fold increases in Nm23-M2 protein expression in HCC (5 month-old mice) as compared with expression in preneoplastic livers (2-month-old mice) from NM23-M1+/+ and NM23-M1–/– mice, respectively. Quantitation relative to normal control liver was not possible because the signal in normal liver was barely detectable. Quantitative real-time RT-PCR corroborated these findings (data not shown). Nm23-M1 and Nm23-M2 protein expression was heterogeneous in HCC lysates from 6-month-old ASV/NM23-M1+/+ mice, as was Nm23-M2 protein expression in HCC from 6-month-old ASV/NM23-M1–/– mice (Fig. 2, B).



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Fig. 2. Nm23 protein expression in ASV mouse liver extracts. Liver tissue homogenates were prepared from mice at different stages of ASV-induced hepatocarcinogenesis and analyzed for Nm23-M1 and Nm23-M2 protein expression by Western blotting with polyclonal anti-Nm23-H1 and anti-Nm23-H2 antibodies, respectively. A) Nm23 expression in livers from 2-, 3-, and 5-month-old ASVNM23-M1+/+ (+/+) and ASV/NM23-M1–/– (–/–) male mice and in normal liver from a 3-month-old female mouse that did not express simian virus (SV40) and did not develop hepatocellular carcinoma (HCC) (C). The liver samples from the 5-month-old mice were HCCs. B) Nm23 protein expression in HCC from seven ASV/NM23-M1+/+ (lanes 1 to 7) and seven ASV/NM23-M1–/– (lanes 8 to 14) 6-month-old male mice. All blots were reprobed with an anti-extracellular signal–regulated kinase (ERK) antibody (which detects both ERK1 and ERK2) to ensure equivalent protein loading.

 
Hepatic NDPK Activity in Diethylnitrosamine-Treated and ASV Mice

We measured total NDPK activity in liver lysates from diethylnitrosamine-treated and ASV mice. In both models, normal liver (from untreated 6-month-old male mice and from 3 month-old-female mice, in the diethylnitrosamine and ASV models, respectively) from NM23-M1–/– mice exhibited almost twofold lower NDPK activity than that from NM23-M1+/+ mice (P<.001) (Table 3). This difference persisted during HCC development, suggesting that Nm23-M1 contributed markedly to liver NDPK activity. HCC extracts from diethylnitrosamine-treated NM23-M1+/+ and NM23-M1–/– mice had statistically significantly higher NDPK activity than extracts of nontumor liver tissue (P = .015) (Table 3). The slight increase in NDPK activity observed in HCC relative to normal liver in both ASV/NM23-M1+/+ and ASV/NM23-M1–/– mice was not statistically significant (P = .096) (Table 3).


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Table 3.  Mean hepatic nucleoside diphosphate kinase (NDPK) activity (95% confidence interval) in diethylnitrosamine-treated mice and ASV mice*

 
Characterization of Lung Nodules in Diethylnitrosamine-Treated and ASV Mice

We observed nodules in the lungs of diethylnitrosamine-treated mice and ASV mice. To assess the metastatic origin of the lung nodules, we performed histologic studies and an immunohistochemical analysis of the specific hepatocyte marker Hep-Par-1 using sections of liver and lung tissue. In diethylnitrosamine-treated mice, hepatic tumor tissue was positive for Hep-Par 1 (Fig. 3, A), whereas lung nodules were negative (Fig. 3, C), suggesting that the latter were primary tumors induced by diethylnitrosamine. In addition, the lung nodules bore no histologic resemblance to hepatic tumor cells, but rather histologically resembled bronchioloalveolar adenomas. In ASV mice, lung nodules were observed at 6 months of age and were morphologically similar to HCC. These nodules, like HCC (Fig. 3, B), were positive for Hep-Par 1 (Fig. 3, D), confirming their metastatic origin.



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Fig. 3. Immunohistochemical analysis of liver and lung sections of diethylnitrosamine-treated and ASV mice with Hep-Par 1 antibody, which recognizes an antigen in hepatocyte mitochondria. Liver and lung sections of NM23-M1–/– mice were immunostained with the Hep-Par 1 antibody. Reddish-brown corresponds to Hep-Par 1 antibody binding and blue corresponds to hematoxylin counterstaining. A) Liver section with a Hep-Par 1–positive hepatocellular carcinoma (HCC) from a 12-month-old diethylnitrosamine-treated NM23-M1–/– mouse. B) Liver section with a Hep-Par 1–positive HCC from a 6-month-old ASV/NM23-M1–/– mouse. C) Lung section with a Hep-Par 1–negative bronchioloalveolar adenoma from a 12-month-old diethylnitrosamine-treated NM23-M1–/– mouse. D) Lung section with a Hep-Par 1–positive metastasis from a 6-month-old ASV/NM23-M1–/– mouse. Scale, 100 µm.

 
Metastatic Potential in ASV Mice

We next examined the effect of a lack of the NM23-M1 gene on HCC metastasis to the lung in ASV mice. We evaluated the number of mice that developed lung metastases as well as the number of metastases per mouse (Table 4). Statistically significantly more ASV/NM23-M1–/– mice than ASV/NM23-M1+/+ mice developed lung metastases (69.2% versus 37.5%; difference = 31.7%, 95% CI = 13.1% to 50.3%; P<.001). The incidence of lung metastases in ASV/NM23-M1+/– mice was also statistically significantly higher than that in ASV/NM23-M1+/+ mice (58.6% versus 37.5%; difference = 21.1%, 95% CI = 3.0% to 39.0%; P = .013), indicating a gene dosage effect (Table 4). However, among ASV mice with metastases, the number of metastases per mouse and the size distribution of metastatic foci were similar for NM23-M1+/+ and NM23-M1–/– mice (Table 4).


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Table 4.  Analysis of metastatic pulmonary dissemination in ASV mice*

 
Nm23-M1 Protein Expression in Liver Tumors and Lung Metastases of ASV/NM23-M1+/+ Mice

We analyzed Nm23-M1 protein expression in liver tumors and lung metastases of ASV mice by using immunohistochemistry. We observed heterogeneous antibody labeling among different tumoral liver tissue samples (Fig. 4, A, a and b) as well as within individual tumoral liver tissue samples (Fig. 4, A, c), a finding that suggests that some clones of hepatic tumor cells had lost Nm23-M1 expression, an event that could favor their metastatic spread. Among approximately 300 lung metastases from ASV/NM23-M1+/+ mice examined, 78% exhibited no antibody labeling (Fig. 4, A, d) and 22% exhibited weak or moderate labeling (Fig. 4, A, e). We hypothesized that NM23-M1+/+ mice presenting with metastases could have lost Nm23-M1 protein expression in the primary tumor. We therefore selected three groups of mice which had distinctly different numbers of pulmonary metastases [no metastases, a low number of metastases (10–15), and the highest number of metastases (50–150)] and analyzed HCC samples from these mice for the presence of Nm23-M1 by Western blotting (Fig. 4, B). In accordance with this hypothesis, mice with the highest number of metastases had the lowest levels of Nm23-M1 protein. Quantitation of the data by densitometry scanning showed a mean 3-fold lower [95% CI = 1.8-fold to 5.5-fold] Nm23-M1 protein expression in HCC from NM23-M1+/+ mice exhibiting the highest numbers of metastases as compared with those with no metastases.



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Fig. 4. Nm23-M1 expression in hepatocellular carcinoma (HCC) and metastases from ASV/NM23-M1+/+ mice. A) Immunohistochemical analysis of liver and lung sections from a representative mouse with metastases. Reddish-brown corresponds to anti-Nm23-M1 antibody binding and blue corresponds to hematoxylin counterstaining. Examples of liver (a–c) and lung (d and e) sections immunostained for Nm23-M1: a) HCC with no Nm23-M1 protein expression; b) HCC with homogenous Nm23-M1 protein expression in tumor cells; c) HCC with heterogeneous Nm23-M1 protein expression in tumor cells; d) lung metastasis with no Nm23-M1 protein expression; and e) lung metastasis with weak heterogeneous Nm23-M1 protein expression. Scale, 100 µm. B) Western blotting of HCC protein extracts from mice with no metastases (0), mice with 50–150 metastases (+++), and mice with 10–15 metastases (+). Blots were reprobed with anti-extracellular signal–regulated kinase (ERK) antibody to ensure equivalent protein loading.

 
Experimental Metastasis Assay

We next used an experimental metastasis assay to examine whether a lack of the NM23-M1 gene influenced the formation of distant metastases in the lung. NM23-M1+/+ (n = 13) and NM23-M1–/– (n = 11) mice injected intravenously with the metastatic melanoma cell line B16F10 developed similar mean numbers of pulmonary nodules (NM23-M1+/+ mice: 7.8 lung nodules [95% CI = 3.8 to 11.9 lung nodules]; NM23-M1–/– mice: 6.7 lung nodules [95% CI = 4.0 to 9.5 lung nodules]). The maximal diameters of these nodules were similar in the two groups and were mostly less than 0.3 mm (data not shown).

Cyclin A Protein Levels in ASV/NM23-M1–/– Mice

We examined the effect of the lack of the NM23-M1 gene on the expression of different markers of tumor progression in ASV mice by Western blot analysis. We observed no difference in c-Myc or Rho A protein level or in ERK1/2 phosphorylation between ASV/NM23-M1+/+ mice and ASV/NM23-M1–/– mice (data not shown). The Rho C isoform was not detected in either group of mice. Cyclin A levels were slightly increased in preneoplastic lesions compared with normal liver from female mice and were markedly increased in HCC from 5- and 6-month-old ASV/NM23-M1+/+ mice (Fig. 5) as compared with preneoplastic stages. By contrast, ASV/NM23-M1–/– mice of all ages displayed low cyclin A levels. Quantitation of the data by densitometry scanning showed a mean 3.5-fold (95% CI = 2.9-fold to 3.8-fold) increase in cyclin A protein expression in HCC compared with that in preneoplastic livers from NM23-M1+/+ mice, but no increase in HCC from NM23-M1–/– mice. Quantitation relative to normal control liver was not possible because the cyclin A signal is null in normal liver was barely detectable. Expression of cyclin D1, cyclin E, and the cyclin A regulators, Cdk2 and p21, increased in ASV/NM23-M1+/+ and ASV/NM23-M1–/– mice during HCC development, with no substantial differences between the two groups of mice (data not shown).



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Fig. 5. Cyclin A expression in ASV mice. Cyclin A protein expression was analyzed by Western blotting of liver tissue homogenates from ASV/NM23-M1+/+ (+/+) and ASV/NM23-M1–/– (–/–) mice during tumor development. C = a control female mouse that did not develop hepatocellular carcinoma. Blots were reprobed with anti-extracellular signal–regulated kinase (ERK) antibody to ensure equivalent protein loading.

 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We examined the role of NM23 in tumor development and metastatic dissemination by using HCC-prone transgenic mice lacking NM23-M1, the gene that encodes the A subunit of NDPK. Because NDPK is responsible for the synthesis of nucleic acid precursors, we expected that tumor proliferative capacity might be affected in mice that lacked genes encoding subunits of NDPK. Indeed, overexpression of Nm23-M1 and/or Nm23-M2 has been reported in lymphocytes induced to proliferate (39) and in various human tumors relative to the corresponding non-tumor tissues (1518). However, in the two HCC models we used in this study, the lack of the NM23-M1 gene had no effect on primary liver tumor formation, the development of preneoplastic liver nodules or overt hepatocellular carcinoma, or the size of the hepatic lesions. In the diethylnitrosamine model, NM23-M1+/+ mice displayed a marked increase in the expression of Nm23-M1 (and, to a lesser extent, of Nm23-M2) in neoplastic lesions compared with nontumor liver tissue. However, in ASV/NM23-M1+/+ mice, only Nm23-M2 was overexpressed in HCC compared with normal liver during hepatocellular carcinogenesis. In the ASV/NM23-M1+/+ mice, some liver tumors had a diminished Nm23-M1 content relative to liver tumors of other mice of the same age and genotype. Together, these data indicate that overexpression of Nm23-M1 and/or Nm23-M2 is related to hepatocellular carcinogenesis and that the two Nm23 isoforms may have redundant functions during primary tumor development. The increases in Nm23-M1 and/or Nm23-M2 levels that we observed were not associated with marked increased in NDPK activity, suggesting that another factor(s) may be involved in controlling NDPK activity, as has been previously reported (12).

The incidence of lung metastases (the number of mice with metastases) in ASV/NM23-M1–/– mice was almost twice that in ASV/NM23-M1+/+ mice. Because the lack of the NM23-M1 gene had no influence on primary tumor formation but did influence metastatic spread, these two processes appear to be independent, as previously suggested by studies of other metastasis suppressor genes (40,41). Surprisingly, ASV/NM23-M1+/+ mice and ASV/NM23-M1–/– mice developed similar numbers of metastatic lung nodules per mouse, and the nodules were of similar size. One possible explanation for these apparently contradictory observations is that the metastatic process in ASV/NM23-M1+/+ mice is accompanied by a loss of Nm23-M1 protein expression in both the primary tumor and the metastases. In support of this possibility, we found that the primary tumors displayed highly heterogeneous labeling with an antibody to Nm23-M1. It is possible that the loss of Nm23-M1 protein expression in some tumor cells might enhance their capacity for metastatic spread. In addition, our finding that most lung metastases did not label with the anti–Nm23-M1 antibody suggests that metastatic dissemination of HCC in ASV/NM23-M1+/+ mice was associated with decreased expression of Nm23-M1. Our Western blot analyses showed that ASV/NM23-M1+/+ mice with the most lung metastases per animal had lower Nm23-M1 expression in HCC than ASV/NM23-M1+/+ mice with the fewest lung metastases per animal. The heterogeneous Nm23 labeling of the primary tumor, which has also been seen in human tumors (17), together with the use of antibodies and probes that did not discriminate between the closely related isoforms, may have undermined previous studies aiming to examine the association between tumor NM23-H1 expression and tumor aggressiveness (42,43).

In the experimental metastasis assay (i.e., intravenous injection of B16F10 murine melanoma cells), the number and size of metastatic nodules were similar in NM23-M1+/+ mice and NM23-M1–/– mice, indicating that the lack of NM23-M1 gene expression in the target organ (the lung) neither induced selective homing of tumor cells to this organ nor conferred a selective advantage on them for tumor proliferation, as proposed in the "seed and soil" hypothesis (44). This finding suggests that NM23 gene products may instead have direct effects on the intrinsic properties of tumor cells. Results of previous transfection studies have shown that overexpression of the NM23 gene in several metastatic cell lines reduced the metastatic potential of the tumor cells in in vitro migration assays and in in vivo experimental metastasis assays (8,11).

The biochemical mechanisms underlying the metastasis-suppressing action of NM23-M1 are largely unknown. Nm23 proteins are multifunctional, and they interact with many different proteins, some of which are involved in cancer progression [reviewed in (45)]. Nm23 proteins also bind and cleave DNA [reviewed in (31)], and they regulate several promoters, including those in the c-myc (30,31) and PDGF-A genes (28). However, we observed no difference between ASV/NM23-M1+/+ mice and ASV/NM23-M1–/– mice with respect to the level of c-Myc protein in liver extracts or PDGF-A mRNA levels (Boissan M, Munier A, Lacombe ML: unpublished observations). It has also been suggested that NDPK could locally modulate the GTP pool and thereby influence signal transduction (20,21). Interactions between Nm23-M1 and G proteins have been reported (23,4648). Although we observed no difference between ASV/NM23-M1+/+ mice and ASV/NM23-M1–/– mice with respect to their levels of the Rho A protein, a member of the Rho GTPase family, we did not examine the activation status of this protein or that of other Rho family members that have been implicated in tumor aggressiveness (49,50). Nm23-H1, the human homolog of Nm23-M1, has also been reported to co-immunoprecipitate with KSR, a scaffold protein that functions downstream of RAS and is required for the ERK1/2–MAP kinase activation pathway (26), which is involved in tumor invasion (51). A breast carcinoma cell line overexpressing Nm23-H1 protein displayed reduced ERK1/2 activation, i.e., reduced levels of phosphorylated ERK1/2 when compared to untransfected cell line (26). We found that the phosphorylation status of ERK1/2 in liver extracts from mice lacking NM23-M1 gene expression was the same as that in liver extracts from mice homozygous for the wild-type NM23-M1 gene (data not shown). Nm23-H1 and abnormal wing discs (awd), the Drosophila ortholog of Nm23-H1 and Nm23-H2, have also been proposed to be activators of the GTPase dynamin that facilitate endocytosis of growth factor receptors, thereby attenuating their signaling (48,52,53). Nm23-H1 and Nm23-H2 also interact with proteins involved in cell movement and adhesion (25,54). Thus, the lack of NM23 expression could maintain or prolong growth factor activity or reduce cell adhesiveness.

We found that in ASV/NM23-M1+/+ mice, cyclin A expression was higher in HCC than in liver at preneoplastic stages, whereas in HCC of ASV/NM23-M1–/– mice the cyclin A level remained low. Several studies have reported an inverse association between cyclin A expression and metastatic dissemination (55,56).

We have focused our study on the role of NM23-M1 on metastatic dissemination in animal models and obtained clear evidence for the involvement of this isoform in metastatic spread. However, we also reported alterations in the expression of the closely related Nm23-M2 isoform. We cannot exclude the possibility that the balance between both isoforms is important in this process. Further analyses of the respective expression of both isoforms in animal and human cancers will be required to clarify the role of NM23-M2 in metastatic spread.

In conclusion, our results provide the first clear evidence that the NM23-M1 gene acts as a suppressor of solid tumor metastasis. In fact, we observed a dual NM23 regulation in wild-type mice: an increase in Nm23-M1 and Nm23-M2 protein expression during primary tumor formation and a decreased expression of Nm23-M1 protein, concomitant with metastatic dissemination in both primary tumors and metastatic nodules. Our work with the NM23-M1 knockout mice demonstrates that the loss of Nm23-M1 protein expression is involved in metastatic spread. In the future, these knockout mice could be useful for establishing models of metastatic dissemination in other cancers. The mechanisms by which NM23-M1/H1 contributes to the metastatic process remain to be elucidated.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This work was supported by grants from Institut National de la Santé et de la Recherche Médicale (INSERM) and from the Groupement des Entreprises Françaises dans la Lutte contre le Cancer (GEFLUC), by grants (5911 to M.-L.L.) from the Association pour la Recherche contre le Cancer (ARC) and by fellowships (to M.B.) from the Fondation pour la Recherche Médicale (FRM) and from ARC.

We are very grateful to Dr. P. Briand for the gift of ASV mice, to Dr. M. F. Poupon for the gift of the B16F10 cell line and helpful advice, to Prof. J. Capeau and to Dr. C. Desbois-Mouthon for helpful comments and critical reading of the manuscript, and to C. Rey for help with animal surgery.


    REFERENCES
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Manuscript received November 19, 2004; revised March 22, 2005; accepted April 8, 2005.



             
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