Nm23-H1

Nonmetastatic protein 23
Nucleoside diphosphate (NDP) kinase-A (NDPKA) (Gilles A.-M. et al., 1991)
NME1
NM23-1, nm23-1

Gene: maps to 17q22 (Varesco L. et al., 1992) and consists of 5 exons and 4 introns spanning 8.5 kb (Dooley S. et al., 1994). The gene is separated from the nm23-H2 gene by no more than 18 kb. The 2 genes may have arisen by a tandem duplication. The expression of the nm23-H1 gene is related to cell proliferative activity (Keim D. et al., 1992). The nm23-H1 promoter has no TATA box, but it contains a number of sequences which may bind known transcriptional regulatory proteins (AP-1, CTF/NF1, ACAAAG, and Ets). Two nonconsensus transcriptional start sites within one of the Ets binding sites. Nuclear proteins from HeLa cells bound specifically to a 95 bp region of the nm23-H1 promoter which harbors the CTF/NF1 recognition consensus sequence, suggesting that CTF/NF1 may play a role in nm23-H1 expression (de la Rosa A. et al., 1996).
mRNA: size: 0.8 kb. At cDNA level, nm23-H1 is 88% identical to nm23-H2.
Protein: ~17 kD. Amino acid differences between nm23-H1 and nm23-H2 are highly conservative in hydrophobicity and charge, bringing the total homology of the two proteins to 98%.
Nm23 is a heterodimeric protein which acts as a nucleoside diphosphate (NDP) kinase (Gilles et al., 1991). Nm23-H1 and nm23-H2 encode the A and B polypeptide chains of the enzyme. Each chain consists of 152 amino acid residues. NDP kinases are involved in the synthesis of nucleoside triphosphates, and the nm23 protein may act in the regulation of signal transduction by complexing with G proteins, causing activation/inactivation of developmental pathways.

Cell lines:
- Both nm23-H1 and nm23-H2 mRNA levels were reduced in the estrogen-independent, metastatic MDA-MB-435 cell line as compared to the estrogen-dependent, non-metastatic MCF-7 cell line. Densitometric analysis of multiple blots indicated that nm23-H1 mRNa exhibited a 3- to 4- fold decrease, while only a 20% decrease was noted for nm23-H2 (Stahl J.A. et al., 1991).

- MDA-MB-435 human breast cancer cells (BCC) exhibited reduced response to motility-stimulating factors following nm23-H1 transfection (Kantor J.D. et al., 1993).

- MCF-7 BCC line was found to express four- and tenfold higher levels of nm23-H1 than the highly metastatic MDA-MB-435 and MDA-MB-231 BCC, respectively. Among the cell lines, there was an inverse correlation between nm23-H1 expression and metastatic potential in athymic nude mice. The differences between the levels of nm23-H1 among the metastatic and nonmetastatic cell lines were, however, not statistically significant (Russell R.L. et al., 1997).

- The nm23-H1 gene was transfected into MDA-MB-231 BCC and 12 clones were selected. No significant differences were found in cathepsin D, uPA, PAI-1 or uPAR, as a function of nm23-H1 expression in either the MDA-MB-231 BCC or the transfected clones. However, compared with the parent cell line, two clones with four- and eightfold elevated nm23-H1 expression showed a dose-dependent decrease in growth factor stimulated motility and a decrease in metastatic potential, whereas the proliferative activities were similar. The decreased metastatic potential might be related to down-regulation of growth factor-stimulated motility (Russell R.L. et al., 1998).

- Mutations of either proline-96 to serine or serine-120 to glycine in nm23-H1 caused MDA-MB-435-wtH1-transfected BCC to revert to parental levels of motility responsiveness to 0.5% serum or autotaxin. the analyses of purified nm23 mutants of proline-96 or serine-120 demonstrated alterations in autophosphorylation and histidine kinase activity (MacDonald N.J. et al., 1996; Freije J.M.P. et al., 1997).

- The distribution of the A (nm23-H1) and B (nm23-H2) NDP kinases was analyzed by immunocytofluorescence microscopy in human BCC lines (MCF-7 and MDA-MB-231) using highly specific polyclonal and monoclonal antibodies. Interphasic cells exhibited a granular and filamentous cytoplasmic staining, particularly intense around nuclei, with both anti-NDP kinase A (nm23-H1) and B (nm23-H2) antibodies. The filamentous component observed with either anti-A (nm23-H1) or anti-B (nm23-H2) antibodies was altered in parallel to tubulin labeling with compounds interacting with microtubules, such as taxol and colchicine. Confirming published biochemical data, a partial colocalization with the vimentin network was observed in the MDA-MB-231 cell line. A nuclear and nucleolar localization of NDP kinase B (nm23-H2) was shown by confocal microscopy which was not observed with the A (nm23-H1) enzyme. In dividing cells, NDP kinase labeling was punctiform and was not colocalized with the mitotic spindle. In conclusion, the A (nm23-H1) and B (nm23-H2) NDP kinases are similarly distributed in cytosol, associated partly to microtubules supporting a role in nucleotide channeling. Only the B (nm23-H2) enzyme is present in nuclei in accord with its role as a DNA binding protein. Their altered localization in dividing cells suggests colocalization with yet unidentified structures which are not intermediate filament aggregates (Pinon V.P. et al., 1999).

Tumors:
- Levels of nm23-H1 protein or RNA have shown an inverse correlation with lymph node status and patient survival in a number of human breast cancer studies (Bevilacqua G. et al., 1989; Barnes R. et al., 1991; Hennessy C. et al., 1991; Hirayama R. et al., 1991; Royds J.A. et al., 1993).

- The relationship between the expression of nm23 protein and the metastatic potential of human breast carcinoma was analyzed in cell lines, xenografts, and in a retrospective lymph node negative breast carcinoma population. The lymph node negative breast carcinoma study was comprised of 40 patients: 19 with nonrecurrent and 21 with recurrent disease. The 40 patients were matched according to age, cathepsin D, tumor size, percent S-phase, DNA ploidy, steroid receptor status, and tumor grade. Statistical analyses indicated that neither the intensity nor the percent of tumor staining positive for nm23 expression was correlated to the recurrence of breast carcinoma in the lymph node negative patient population that had been matched for other clinical prognostic markers (Russell R.L. et al., 1997).

- In a study involving 152 breast cancer samples, there was no significant relationship between either of the nm23 isoforms (nm23-H1, nm23-H2) and cathepsin D, uPA, PAI-1, steroid hormone receptors (ER, PgR) or ploidy status. However, a positive correlation was observed between uPA receptor and both nm23-H1 (P=0.0018) and nm23-H2 (P=0.0064) (Russell R.L. et al., 1998).

- To evaluate the significance of nm23 and angiogenesis in the metastatic cascade, archival material from 163 node-negative breast cancer patients who had a median follow-up of 14 years was studied. All patients underwent mastectomy and received no adjuvant chemotherapy or hormone or radiation therapy. Immunohistochemistry was used to detect nm23-H1 expression, whereas angiogenesis was determined by microvessel count (MVC). 15-year disease-free survival (DFS) was found to be significantly better in patients with high nm23 compared with low nm23 (91% compared with 70%, P = 0.008). Low MVC was associated with excellent (92%) long-term DFS. In those patients with high MVC, high nm23 allowed the identification of a subgroup with significantly higher DFS (90% compared with 66%, P = 0.02). Among high nuclear grade tumors, if nm23 was high, the DFS was significantly better (89% compared with 68%, P = 0.03). Thus, nm23 was still associated with excellent survival, even when there was unfavorable angiogenesis or nuclear grade. Multivariate analysis confirmed that nm23 and MVC were important prognostic factors. High MVC appeared necessary but not sufficient for metastasis to occur, whereas low nm23 may further contribute to metastatic progression (Heimann R. et al., 1998).

- 102 surgically resected primary breast cancer tissues were sectioned and stained with antibody against nm23-H1 and Sialyl Lewis X (sLex), which has been demonstrated to play an important role in the adhesion of human cancer cells to human vascular endothelium. Of the 102 cases, 39 (38.2%) cases with a reduced expression of nm23-H1 were observed, and the numbers of sLex-positive cases were 61 (59.8%), respectively. The reduced expression of nm23-H1 and the positive expression of sLex were significantly associated with lymph node involvement. Among the 100 patients who underwent curative surgery, the disease-free survival rate was significantly correlated to both the nm23-H1 and sLex expressions. No interrelated expressions were found between nm23-H1 and sLex (Yamaguchi A. et al., 1998).

- Nm23-H1 immunocytochemical assays (ICAs) were performed on frozen sections, using an automated immunoperoxidase technique and computer-assisted analysis of digitized colour microscopic images, in a series of 168 breast carcinomas. The results of automated quantitative ICAs were correlated with patients' follow-up (129 months). Nm23-H1 immunocytochemical expression in histological sections of tumours in which more than 3 per cent of the surface area was positively stained was significantly correlated with longer metastasis-free survival in both node-positive and node-negative groups of patients. Nm23 expression (cut-point 3 per cent) did not, however, correlate with overall survival, or with the recurrence-free survival. It was concluded that Nm23-H1 immunodetection is only of limited practical clinical relevance in breast carcinoma, even when assessed under optimal technical conditions (Charpin et al., 1998).

- The expression of the nm23-H1 gene peptide was immunohistochemically evaluated in 191 primary mammary cancer tissues. nm23-positive cytoplasmic immunolabeling was noticed in 64% of all specimens (123/191) which frequently demonstrated positive progesterone receptor (PgR) status and were furthermore characterized by high PgR immunoreactivity rates. This association was significant by both univariate and multivariate statistical analysis. The double nm23-H1 (+)/PgR(+) phenotype was more frequently detected than any other combined phenotype of these markers. The nm23-H1 gene peptide was generally detected in a remarkable proportion of malignant cells, either in the invasive or the intraductal tumor components. Notably, large-cell ductal carcinomas in situ were characterized by lower nm23-H1 immunoreactivity rates when compared with other in situ cancer types. Quantitatively increased nm23-H1 immunopositive staining was more frequently observed in special histologic types of infiltrating cancers, in high nuclear grade tumors, as well as in carcinomas with high PgR levels. The nm23-H1 (-)/c-erbB-2(+) phenotype was more often detected in the cancers of this study than the nm23-H1(+)/c-erbB-2(+) one. The former phenotype was correlated to postmenopausal ages as well as to extensive axillary nodal involvement by univariate statistical analysis. It is noteworthy that nm23-H1(-) status, on its own, was not statistically associated either with the presence or with a high number of involved lymph nodes. On the contrary, nm23-H1 immunopositivity was, paradoxically, more frequently observed in tumors of relatively increased TN tumor stage. Tumor progression is thus more likely to depend on the c-erbB-2 gene's overexpression. Possibly, any favorable outcome in nm23-H1(+) cases might be due to the fact that they also express PgR, which is a marker of a more functionally differentiated phenotype (Nakopoulou L.L. et al., 1999).

Barnes R. et al. (1991) Low nm23 protein expression in infiltrating ductal breast carcinomas correlates with reduced patient survival. Am. J. Pathol. 139, 245-250.
Bevilacqua G. et al. (1989) Association of low nm23 RNA levels in human primary infiltrating ductal breast carcinomas with lymph node involvement and other histopathological indicators of high metastatic potential. Cancer Res. 49, 5185-5190.
Charpin C. et al. (1998) Prognostic significance of Nm23/NDPK expression in breast carcinoma, assessed on 10-year follow-up by automated and quantitative immunocytochemical assays. J. Pathol. 184, 401-407.
de la Rosa A. et al. (1996) Identification and characterization of the promoter for the human metastasis suppressor gene nm23-H1. Arch. Med. Res. 27, 395-401.
Dooley S. et al. (1994) Isolation and characterization of the human genomic locus coding for the putative metastasis control gene nm23-H1. Hum. Genet. 93, 63-66.
Freije J.M.P. et al. (1997) Site-directed mutation of Nm23-H1. J. Biol. Chem. 272, 5525-5532.
Gilles A.-M. et al. (1991) Nucleoside diphosphate kinase from human erythrocytes: structural characterization of the two polypeptide chains responsible for heterogeneity of the hexameric enzyme. J. Biol. Chem. 266, 8784-8789.
Heimann R. et al. (1998) The relationship between nm23, angiogenesis, and the metastatic proclivity of node-negative breast cancer. Cancer Res. 58, 2766-2771.
Hennessy C. et al. (1991) Expression of the antimetastatic gene nm23 in human breast cancer: an association with good prognosis. J. Natl. Cancer Inst. 83, 281-285.
Hirayama R. et al. (1991) Positive relationship between expression of anti-metastatic factor (nm23 gene product or nucleoside diphosphate kinase) and good prognosis in human breast cancer. J. Natl. Cancer Inst. 82, 1249-1250.
Kantor J.D. et al. (1993) Inhibition of motility after nm23 transfection of human and murine tumor cells. Cancer Res. 53, 1971-1973.
Keim D. et al. (1992) Proliferation-related expression of p19/nm23 nucleoside diphosphate kinase. J. Clin. Invest. 89, 919-924.
MacDonald N.J. et al. (1996) Site-directed mutagenesis of nm23-H1. J. Biol. Chem. 271, 25107-25116.
Nakopoulou L.L. et al. (1999) Nm-23, c-erbB-2, and progesterone receptor expression in invasive breast cancer: correlation with clinicopathologic parameters. Cancer Detect. Prev. 23, 297-308.
Pinon V.P. et al. (1999) Cytoskeletal association of the A and B nucleoside diphosphate kinases of interphasic but not mitotic human carcinoma cell lines: specific nuclear localization of the B subunit. Exp. Cell Res. 246, 355-367.
Royds J.A. et al. (1993) Nm23 protein expression in ductal in situ and invasive human breast carcinoma. J. Natl. Cancer Inst. 85, 727-731.
Russell R.L. et al. (1997) nm23--relationship to the metastatic potential of breast carcinoma cell lines, primary human xenografts, and lymph node negative breast carcinoma patients. Cancer 79, 1158-1165.
Russell R.L. et al. (1998) Relationship of nm23 to proteolytic factors, proliferation and motility in breast cancer tissues and cell lines. Br. J. Cancer 78, 710-717.
Steeg P.S. et al. (1988) Evidence for a novel gene associated with a low tumor metastatic potential. J. Nat. Cancer Inst. 80, 200-204.
Varesco L. et al. (1992) The NM23 gene maps to human chromosome band 17q22 and shows a restriction fragment length polymorphism with BglII. Genes Chromosomes Cancer 4, 84-88.
Yamaguchi A. et al. (1998) Expression of nm23-H1 gene and Sialyl Lewis X antigen in breast cancer. Oncology 55, 357-362.

nm23-H2, DR-nm23