4830

NME1

NME/NM23 nucleoside diphosphate kinase 1;NME1;NME/NM23 nucleoside diphosphate kinase 1

NDP kinase A|granzyme A-activated DNase|metastasis inhibition factor nm23|non-metastatic cells 1, protein (NM23A) expressed in|nucleoside diphosphate kinase A|tumor metastatic process-associated protein

17q21.3

protein-coding

OBJECTIVE: The nm23 gene was initially cloned as a metastasis suppressor gene, but the clinical relevance of nm23 as a metastasis suppressor or prognostic indicator for human cancers remain controversial. To evaluate the role of nm23 protein as a prognostic factor and its role in parathyroid neoplasia, we studied nm23 protein expression by immunohistochemical staining in parathyroid lesions. METHODS: Immunohistochemistry using the avidin-biotin peroxidase complex technique with a polyclonal antibody against the nm23 protein was applied to formalin-fixed, paraffin-embedded tissue specimens obtained from 48 patients. The specimens were collected from 38 patients at the University Health Network, Toronto, Canada and from 10 Saudi patients at the King Abdul-Aziz University Hospital, Jeddah, Kingdom of Saudi Arabia. They included parathyroid carcinomas (5 cases), adenomas (22 cases), hyperplasia (21 cases), and normal parathyroid tissue (10 cases). The immunohistochemistry was completed in 2003 at King Abdul-Aziz University Hospital, Jeddah, KSA and University Health Network, Toronto, Canada. RESULTS: expression of nm23 protein was noted in adenomas and carcinomas as well as in hyperplastic parathyroid glands and there was no significant statistical difference between these groups. Normal parathyroid glands did not show any intense immunoreactivity. CONCLUSION: The results suggest that expression of nm23 in parathyroid lesions is correlated with tumor proliferation rather than suppression of invasion and metastasis. While our data suggest that nm23 may help in the distinction of normal from proliferative parathyroids, these results do not point to nm23 as a reliable prognostic marker in parathyroid lesions.

BACKGROUND: The nm23 gene was first identified from murine K-1735 melanoma cell lines and possesses metastasis-suppressor activity. However, conflicting results concerning the metastasis-suppressor activity of nm23-H1 gene product have been reported in human solid tumors. The significance of nm23-H1 protein in bladder cancer remains to be determined. Therefore, we examined nm23-H1 protein expression immunohistochemically in bladder cancer. METHODS: Formalin-fixed, paraffin-embedded tissue specimens were obtained from 39 patients with primary bladder cancer who had undergone radical cystectomy between 1987 and 1994. The specimens were examined immunohistochemically using a monoclonal antibody to nm23-H1, and the results of immunostaining were compared with clinicopathologic factors and patient survival. RESULTS: Positive nm23-H1 protein expression was confined primarily to the cytoplasm of bladder cancer cells. A higher frequency of nm23-H1 positive expression was seen in higher stage tumors. There was a positive trend for the expression of nm23-H1 protein with the progression of the tumor (p < 0.025). No relationship was found between nm23-H1 protein expression and patients' clinicopathologic factors including age, tumor size, tumor morphology and tumor grade. Furthermore, nm23-H1 protein expression was not correlated with patient survival. CONCLUSIONS: These results suggest that in bladder cancer nm23-H1 protein expression may play an important role in tumor progression rather than in metastasis suppression.

The nm23 gene was initially cloned as a metastasis suppressor gene, but the clinical relevance of nm23-H1 as a metastasis suppressor or prognostic indicator for human cancers remains enigmatic. Given that gene expression is regulated at the tissue-specific level, we studied the molecular mechanisms of nm23-H1 expression in human bladder cancer cell lines and the clinical importance of protein product (NM23-H1) in association with patient outcome (n = 257) by immunohistochemistry. We demonstrated that nm23-H1 is expressed in bladder cancer cells without genomic alterations. High NM23-H1 expression was found in 39 cases (15.2%), intermediate expression in 119 cases (46.3%), and low NM23-H1 in 99 cases (38.5%). NM23-H1 was inversely related to staging classification or tumor size (P < 0.05), with the most significant difference being observed between pTa tumors and those of pT1-pT3 bladder cancer (P = 0.01). Reduced NM23-H1, defined as intermediate and low levels of expression, tended to have a higher risk of tumor metastasis (P = 0.06) or poor longtime survival (P = 0.07). In the subset of grade 2 bladder tumors, reduced NM23-H1 significantly correlated with the occurrence of tumor metastasis or poor patient survival (P < 0.05). These findings overall suggest that nm23-H1 may play an important role in suppressing the early step of carcinogenesis and thus act as an invasion suppressor for human bladder cancer. A prospective study is required to clarify the potential of the molecular marker in prediction of disease progression.

Nucleoside diphosphate kinase A (NDPK-A), encoded by the nm23-H1 gene, acts as a metastasis suppressor in certain human tumors such as breast carcinoma. However, evidence also points to NDPK-A functioning as a metastasis promoter in other human tumors including neuroblastoma. In fact, amplification and overexpression of nm23-H1 as well as S120G mutation of NDPK-A (NDPK-A(S120G)) have been detected in 14% to 30% of patients with advanced stages of neuroblastoma. To test whether NDPK-A promotes neuroblastoma metastasis, we established stable transfectants and an orthotopic xenograft animal model from the human neuroblastoma NB69 cell line. We demonstrate that overexpressed NDPK-A or NDPK-A(S120G) increased both incidence and colonization of neuroblastoma metastasis in animal lungs without significantly affecting primary tumor development. In vitro, these metastasis-associated NDPK-A aberrations abrogated retinoic acid-induced neuronal differentiation while increasing cloning efficiency, cell survival, and colony formation of NB69 derivatives. Furthermore, NDPK-A(S120G) reduced cell adhesion and increased cell migration. Compared with its wild-type, NDPK-A(S120G) appears more effective in promoting neuroblastoma metastasis. Our results provide the first evidence that NDPK-A behaves as a metastasis promoter at least in human neuroblastoma derived from NB69 cells. The findings not only suggest a prognostic value of NDPK-A in neuroblastoma patients but also caution NDPK-A-targeted treatment for patients with different tumor types.

Data obtained in experimental murine tumors and in clinical specimens of human breast cancer have suggested that the nm23 gene may function as a metastasis suppressor gene. In this report we examined the nm23 mRNA level in tumor tissue obtained from distant metastases in 33 patients with malignant melanoma. The gene was differentially expressed in the tumors with a 20-fold range in hybridization intensities. The levels of nm23 mRNA in benign nevi obtained from 12 of the 33 patients were relatively low, with a mean value of 17% of that in the melanomas. In attempts to relate the level of nm23 expression in the tumor metastases to progression of the disease, the time from biopsy of the primary tumor to the appearance of metastases was used as a clinical end point. It was found that patients developing metastases during the first 2 years after diagnosis had significantly lower levels of tumor nm23 expression (56% of the mean value) compared to patients with less aggressive disease (164%) (P < 0.0004). In concordance with previous data the association found here between low levels of nm23 mRNA and the malignant potential of melanomas suggests that the nm23 gene may be implicated in the mechanism of disease progression in some types of human cancer.

Allelic deletions in the nm23, a metastasis suppressor gene, are known to occur in neuroblastomas, breast and colorectal carcinomas. Down-regulation of nm23 expression has been reported in various rodent and human tumor cells with high metastasis phenotype. Colorectal tumors showed overexpression of nm23. To elucidate the regulatory mechanisms of nm23, we isolated, cloned and sequenced the presumptive regulatory DNA fragment spanning the 5' region of the human nm23-H1 gene. The region's nucleotide sequence shows the presence of motifs typical for transcriptional elements such as TFIID, AP-1 and CTF/NF1. A common transcription initiation site is located at -136 upstream from the first ATG codon in placenta tissue, in breast, colorectal, prostate tumor cell lines and in primary colorectal tumor. Multiple transcription start sites were identified in tumor cell lines and colorectal tumor. When the promoter element was linked to a reporter gene, chloramphenicol acetyltransferase (CAT) and transfected in human 2fTGH cells, strong CAT activity was detected, which also showed that the presence of AP-1 and CTF/NF1 elements are essential for promoter activity. A detailed study of the structure and function of the promoter element of the nm23-H1 gene will help in understanding the regulatory mechanisms of nm23 expression and its role in tumor progression, especially in metastasis.

The nm23 gene is a potential metastasis-suppressor gene originally identified in a murine melanoma line. Several investigators have reported the probable inverse association of nm23 expression with disease prognosis and/or metastasis. Since there are now 2 known isotypes of human nm23, namely nm23-HI and -H2, we immunohistochemically examined expression of these isotypes in human breast-cancer tissues using monoclonal antibodies (MAbs) specific for each isotype protein. We also analyzed expression of c-erbB-2 in the same collection of cancer tissues, in order to examine the significance of nm23 expression in comparison with c-erbB-2 expression. Of 130 tumors from breast-cancer patients, 73 (56%) and 69 (53%) positively expressed nm23-HI and -H2 respectively. expression of c-erbB-2 was positive in 36 (28%). expression of nm23-HI, but not nm23-H2, was inversely associated with lymph-node metastasis (p < 0.01). expression of c-erbB-2 was associated with Tnm stage, tumor size and lymph-node metastasis (p < 0.01, p < 0.05 and p < 0.05 respectively). Overall survival was better (p = 0.014) in patients in whom expression of nm23-HI was positive than in those in whom it was negative. In multivariate analyses using a Cox's proportional-hazards regression model with 9 variables, nm23-HI showed the fourth greatest contribution to patient survival following lymph-node metastasis, Tnm stage and menopausal status. No significant contribution was shown for c-erbB-2 expression. nm23-HI, but not nm23-H2, may perform a role in disease prognosis in addition to its participation in cancer metastasis. It may have value for predicting long-term survival of human breast-cancer patients.

Identification of multiple clinical and pathologic prognostic factors in differentiated thyroid cancer has permitted some degree of risk stratification. However, these clinical indices fail to distinguish potential intrinsic differences in tumor virulence. The nm23 gene has been identified as a potential metastasis suppressor gene that is homologous to nucleoside diphosphate kinases. Studies in human breast cancer have shown a significant inverse correlation between nm23 levels and nodal involvement/tumor recurrence. Given the possible clinical utility of a marker of metastatic potential in the management of thyroid carcinoma, we examined 34 thyroid neoplasms and a human medullary thyroid cancer (MTC) cell line (TT) for nm23 expression. Normalized nm23 expression was assessed by Northern analysis of tumor RNA. nm23 expression (tumor expression/TT cell expression, mean +/- SE) was 1.14 +/- 0.15* in MTCs (n = 5), 0.70 +/- 0.10* in follicular cancers (n = 6), 0.51 +/- 0.11 in papillary cancers (n = 19), and 0.31 +/- 0.03 in follicular adenomas (n = 4) (*p < 0.05 when compared to adenomas). Within histologic groups, we found no correlation between nm23 expression and nodal involvement of distant metastases. Our results indicate that thyroid neoplasms of different histologies express varying levels of the nm23 transcript. Although nm23 expression seems diminished in metastatic breast cancer, it appears not to be the case in metastatic thyroid cancer. The nm23 gene may therefore have different roles in the evolution and metastases of different neoplasms.

BACKGROUND: Mortality associated with human breast carcinoma is almost entirely due to subsequent metastatic disease, but the molecular basis of this metastasis is not understood. Elucidation of the genetic control of metastatic propensity of a tumor is important in determining prognosis and choice of therapy. expression of nm23, a putative metastasis suppressor gene, has been detected in human breast cancers, but studies have not consistently shown high levels of the Nm23 messenger RNA or protein to be associated with better histological differentiation. This inconsistency suggests that Nm23 protein may act independently as a metastasis suppressor. PURPOSE: The purpose of this retrospective study was to investigate the relationship of Nm23 protein expression with 1) histology in ductal breast carcinoma in situ and 2) the variables considered to be the major prognostic indicators in invasive breast carcinoma. METHODS: We obtained formalin-fixed biopsy specimens of breast tissue excised from 128 patients with breast lesions detected by mammography. Of these patients, 35 had been diagnosed with benign breast disease, 26 with ductal carcinoma in situ (DCIS), and 67 with invasive carcinoma. Tissue sections were embedded in paraffin blocks, and immunohistochemical staining was used to determine Nm23 expression. Specimens were rated positive if all lesional epithelium was stained and negative if any lesional epithelium was unstained. Statistical analysis was performed by multiple regression analysis because of nonorthogonality of the data. RESULTS: All 35 examples of benign breast disease showed uniform epithelial cell staining. The seven cases of comedo DCIS were negative for Nm23 protein; all 18 noncomedo types were positive. Nm23 negativity was significantly associated with worsening invasive ductal carcinoma grade and advancing lymph node stage but not with tumor diameter or vascular invasion. Despite the putative antimetastatic role of the nm23 gene, no statistically significant association was found between Nm23 protein expression and vascular invasion. CONCLUSIONS: The precise role of the nm23 gene remains to be established, but our simplified immunohistochemical rating system shows an association between Nm23 protein expression and the two most significant prognostic factors relating to histologic grade and stage. Nm23 negativity distinguished comedo ductal carcinoma in situ from the other histological types, a finding consistent with the fact that comedo histology is known to have a higher likelihood of becoming invasive and of having higher cell proliferation rates and higher expression of growth factor (c-erb B2) receptor.

BACKGROUND: The murine nm23 gene suppresses the metastatic behavior of malignant rodent tumor lines, and reduced nm23 expression correlates with increased likelihood of lymph node metastases in human breast cancers. More recent data have demonstrated the existence of two human nm23 gene homologues, nm23-H1 and nm23-H2, and have shown that deletion of nm23-H1 alleles occurs in some colon carcinomas associated with poor prognosis. These findings suggest that nm23-H1 encodes for suppression of colon carcinoma metastasis. In contrast, we have previously reported that total nm23 messenger RNA (mRNA) expression is increased to similar levels in colon tumors of both high and low metastatic potential. PURPOSE: This study was designed to reconcile our previous findings with the recent report of nm23-H1 allelic deletion in human colon cancers associated with poor prognosis. Our purpose was to examine human colon cancers for inactivation of two candidate metastasis suppressor genes, nm23-H1 and nm23-H2, either by mutation or by loss of gene transcription. METHODS: We used ribonuclease protection assays to analyze human colon tumors for the level of nm23-H1 (43 samples) and nm23-H2 (41 samples) transcript (mRNA) expression and the presence of mutations that could inactivate potential suppressor function. RESULTS: We detected only wild-type nm23-H1 and nm23-H2 mRNA. expression of nm23-H1 mRNA increased in 33 of 41 colon tumors, and expression of nm23-H2 mRNA was elevated in 28 of 41 colon tumors relative to that in matched normal mucosa. Increases in these mRNA levels were similar in tumors of both low and high metastatic potential. CONCLUSIONS: These results suggest that, despite correlation of nm23-H1 allelic deletions with colon cancers associated with poor prognosis, nm23-H1 and nm23-H2 alleles do not directly mediate metastasis suppression in colon carcinoma. Our results leave unexplained the observation that nm23-H1 allelic deletion correlates with metastatic potential of colon carcinomas. IMPLICATIONS: These findings also contrast with the demonstration of nm23 metastasis suppressor activity in murine melanoma and with the correlation of loss of nm23 expression in breast cancer with poor prognosis. It may be that metastasis suppression by the nm23 gene is a tissue-specific phenomenon.

The metastasis-suppressor nme gene (also called nm23), first identified in murine melanoma cells, exists as two forms in human: nme1 and nme2. However, only the lack of expression of nme1 has been related to distant metastasis appearance in human breast cancer. The aim of this work was first to raise specific antibodies to allow the analysis of Nme1 and then, with this specific tool, to evaluate the predictive value of Nme1 detection in cytosolic extracts of human breast tumours. We obtained a hen antibody that specifically reacts with Nme1 without any cross-reaction with Nme2. We analysed the expression of the protein in 59 human breast tumours and found a significant relationship between this expression and oestrogen receptor status (P<0.001). Moreover, Nme1 expression is related to metastasis-free survival (P<0.001) and survival of patients (P<0.001). The determination of Nme1 expression in primary tumours using our antibody should be an interesting predictive test of the metastasis for clinical investigations.

To determine the relevance of genetic information on chromosome 11 in the development of metastatic breast tumors, we introduced a normal human chromosome 11 into the highly metastatic MDA-MB-435 breast carcinoma cell line via the microcell-mediated chromosome transfer technique. Although the MDA-MB-435 recipient cell line and four randomly selected microcell hybrid clones remained tumorigenic in nude mice, the hybrids were >95% suppressed for metastasis to lung and regional lymph nodes (p<0.01). We also tested whether chromosome 6 harbors a metastasis-suppressor gene for breast cancer as observed previously for human melanoma. Grouped together, the four neo6 microcell hybrids had no statistically significant reduction in the incidence or number of lung or lymph node metastases compared to the weakly metastatic, subcloned parent cell line, MDA-MB-453.7. expression of nm23-H1 (NME1), a known metastasis-suppressor gene in this breast cancer cell line, did not correlate with metastasis suppression in the microcell hybrids. These results further demonstrate that control of metastasis is molecularly distinct from tumorigenic potential. They also indicate that chromosome 11 encodes a metastasis-suppressor gene for human breast cancer.

The nm23 gene was originally identified in murine melanoma cell lines as a putative metastasis suppressor gene. 1a a limited number of studies in breast carcinomas nm23 mRNA and/or protein levels were found to correlate inversely with lymph node metastases, and positively with the survival of patients. Using a monoclonal antibody to nm23-Hl protein we have examined the immunohistochemical expression of nm-23 in breast ductal carcinomas of 44 lymph node-negative patients with similar tumor pathologic features. The mean follow-up period of the patients was 138 months. Thirty two out of 44 tumors (72%) disclosed high immunohistochemical expression of nm23 protein and 12 (28%) low or negative expression. No correlation was observed between nm23 expression and the relapse or death rate of the patients. Similarly, no association was found between nm23 protein levels and estrogen receptor status or p53 protein. Our results do not seem to agree with the proposed antimetastatic property of nm23 protein, and indicate that its immunohistochemical determination has no prognose significance in the management of node-negative breast cancer patients.

Nucleoside diphosphate kinase A (NDPK-A), encoded by the nm23-H1 gene, acts as a metastasis suppressor in certain human tumors such as breast carcinoma. However, evidence also points to NDPK-A functioning as a metastasis promoter in other human tumors including neuroblastoma. In fact, amplification and overexpression of nm23-H1 as well as S120G mutation of NDPK-A (NDPK-A(S120G)) have been detected in 14% to 30% of patients with advanced stages of neuroblastoma. To test whether NDPK-A promotes neuroblastoma metastasis, we established stable transfectants and an orthotopic xenograft animal model from the human neuroblastoma NB69 cell line. We demonstrate that overexpressed NDPK-A or NDPK-A(S120G) increased both incidence and colonization of neuroblastoma metastasis in animal lungs without significantly affecting primary tumor development. In vitro, these metastasis-associated NDPK-A aberrations abrogated retinoic acid-induced neuronal differentiation while increasing cloning efficiency, cell survival, and colony formation of NB69 derivatives. Furthermore, NDPK-A(S120G) reduced cell adhesion and increased cell migration. Compared with its wild-type, NDPK-A(S120G) appears more effective in promoting neuroblastoma metastasis. Our results provide the first evidence that NDPK-A behaves as a metastasis promoter at least in human neuroblastoma derived from NB69 cells. The findings not only suggest a prognostic value of NDPK-A in neuroblastoma patients but also caution NDPK-A-targeted treatment for patients with different tumor types.

PURPOSE: Brain metastases are an increasingly common complication in breast cancer patients. The metastasis Suppressor Genes (MSG) Nm23, KISS1, KAI1, BRMS1, and Mkk4 have been associated with the metastatic potential of breast cancer in vitro and in vivo. METHODS: The mRNA expression of Nm23, KISS1, KAI1, BRMS1, and Mkk4 in fresh frozen tissue samples of brain metastases from ductal invasive breast cancer specimens was examined in relation to primary tumors. In a first step, mRNA expression screening was carried out using a semi-quantitative RT-PCR approach, in a second step quantitative real-time RT-PCR was performed on selected specimens. By immunohistochemical staining, gene products were visualized on the protein level. RESULTS: Semi-quantitative RT-PCR revealed reduced mRNA expression of Nm23, KISS1, KAI1, BRMS, and Mkk4 in brain metastases. Results for KISS1, KAI1, BRMS, and Mkk4 were confirmed by real-time RT-PCR. In detail, mRNA expression reduction in breast cancer brain metastases was tenfold. expression of MSG could be confirmed by immunohistochemical staining on protein level. CONCLUSIONS: Our investigations revealed significantly reduced mRNA expression of metastases suppressor genes KISS1, KAI1, BRMS1, and Mkk4 in breast cancer brain metastasis. Particularly, in the case of KISS1 and Mkk4, an important role for future treatment of patients with breast cancer brain metastatic lesions can be assumed.

metastatic disease is a significant contributor to cancer patient mortality. We previously reported that the Kinase Suppressor of Ras1 (KSR1) scaffold protein for the Erk mitogen-activated protein kinase pathway coimmunoprecipitated the metastasis suppressor protein Nm23-H1. We now hypothesize that altered expression levels of Nm23-H1 influence the binding properties, stability, and function of the KSR1 scaffold. Increased coimmunoprecipitation of Hsp90 with KSR1 was observed in either stable or transient transfectants of nm23-H1 in MDA-MB-435 human breast carcinoma cells. Similar trends were also observed in the cytoplasmic and nuclear fractions of cells. Cells expressing high levels of Nm23-H1 exhibited increased KSR1 degradation in the presence of either cycloheximide or an Hsp90-directed drug currently in clinical trial, 17-allylamino-17-demethoxygeldanamycin (17-AAG). In agreement with KSR1 degradation data, high-Nm23-H1-expression cells were preferentially inhibited in anchorage-independent colonization assays by 17-AAG. KSR1 scaffold binding patterns are dynamic in both the cytoplasmic and nuclear compartments, modulated by metastasis suppressor expression. metastasis suppressor expression levels can impact traditional signaling pathways, such as the Erk pathway, resulting in altered tumor cell sensitivity to cancer therapeutics.

BACKGROUND: Reestablishment of metastasis suppressor gene expression may constitute a therapeutic strategy for high-risk breast cancer patients. We previously showed that medroxyprogesterone acetate (MPA), a progestin that has been tested as treatment for advanced breast cancer, elevates expression of the Nm23-H1 metastasis suppressor gene in hormone receptor-negative metastatic human breast carcinoma cell lines in vitro via a glucocorticoid receptor-based mechanism. Here, we tested whether MPA treatment inhibits metastatic colonization of a hormone receptor-negative breast cancer cell line in vivo. METHODS: We tested the soft-agar colony-forming efficiency of untransfected MDA-MB-231T human breast carcinoma cells and MDA-MB-231T cells transfected with antisense Nm23-H1 in the presence and absence of MPA. Pharmacokinetic studies were used to establish dose and injection schedules that led to MPA serum levels in mice similar to those achievable in humans. For in vivo studies, nude mice were injected intravenously with MDA-MB-231T cells. After 4 weeks, mice were randomized to control or MPA arms. Endpoints included incidence, number, and size of gross pulmonary metastases; Nm23-H1 protein expression in gross metastases; and side effects. All statistical tests were two-sided. RESULTS: MPA reduced colony formation of MDA-MB-231T cells by 40%-50% but had no effect on colony formation of Nm23-H1 antisense transfectants. Metastases developed in 100% (95% confidence interval [CI] = 78% to 100% and 77% to 100%, respectively) of control mice injected with MDA-MB-231T cells. In two independent experiments, only 73% (95% CI = 45% to 92%) and 64% (95% CI = 35% to 87%) of mice injected with 2 mg of MPA developed metastases. Mice injected with 2 mg of MPA showed reductions in the mean numbers, per mouse, of all metastases and of large (>3 mm) metastases (P = .04 and .013, respectively). Nm23-H1 was expressed at high levels in 43% of pulmonary metastases in MPA-treated mice but only 13% of metastases in untreated mice. Mice receiving at least 1-mg doses of MPA gained more weight than control-treated mice but exhibited no bone density alterations or abnormal mammary fat pad histology. CONCLUSION: Our preclinical results show that MPA appears to elevate Nm23-H1 metastasis suppressor gene expression, thereby reducing metastatic colonization. The data suggest a new use for an old agent in a molecularly defined subset of breast cancer patients.

Epstein-Barr virus (EBV) is a lymphotrophic herpesvirus infecting most of the world's population. It is associated with a number of human lymphoid and epithelial tumors and lymphoproliferative diseases in immunocompromised patients. A subset of latent EBV antigens is required for immortalization of primary B-lymphocytes. The metastatic suppressor Nm23-H1 which is downregulated in human invasive breast carcinoma reduces the migration and metastatic activity of breast carcinoma cells when expressed from a heterologous promoter. Interestingly, the EBV nuclear antigen 3C (EBNA3C) reverses these activities of Nm23-H1. The alpha V integrins recognize a variety of ligands for signaling and are involved in cell migration and proliferation and also serve as major receptors for extracellular-matrix-mediated cell adhesion and migration. The goal of this study was to determine if Nm23-H1 and EBNA3C can modulate alpha V integrin expression and downstream activities. The results of our studies indicate that Nm23-H1 downregulates alpha V intregrin expression in a dose responsive manner. In contrast, EBNA3C can upregulate alpha V integrin expression. Furthermore, the study showed that the association of the Sp1 and GATA transcription factors with Nm23-H1 is required for modulation of the alpha V integrin activity. Thus, these results suggest a direct correlation between the alpha V integrin expression and the interaction of Nm23-H1 with EBNA3C.

Nm23-H1 transcriptionally down-regulates expression of the lysophosphatidic acid receptor EDG2 and this down-regulation is critical for Nm23-H1-mediated motility suppression in vitro. We investigated the effect of altered EDG2 expression on Nm23-H1-mediated metastasis suppression in vivo. Clonal MDA-MB-435-derived tumor cell lines transfected with Nm23-H1 together with either a vector control or EDG2 had similar anchorage-dependent and anchorage-independent growth rates in vitro. However, a 45- and 300-fold inhibition of motility and invasion (P < 0.0001), respectively, was observed in Nm23-H1/vector lines, whereas coexpression of EDG2 restored activity to levels observed in the parental line. Using fluorescently labeled cells and ex vivo microscopy, the capacity of these cells to adhere, arrest, extravasate, and survive in the murine lung over a 24-h time course was measured. Only 5% of Nm23-H1/vector-transfected cells were retained in the murine lung 6 h following tail vein injection; coexpression of EDG2 enhanced retention 8- to 13-fold (P < 0.01). In a spontaneous metastasis assay, the primary tumor size of Nm23-H1/vector and Nm23-H1/EDG2 clones was not significantly different. However, restoration of EDG2 expression augmented the incidence of pulmonary metastasis from 51.9% to 90.4% (P = 2.4 x 10(-5)), comparable with parental MDA-MB-435 cells. To determine the relevance of this model system to human breast cancer, a cohort of breast carcinomas was stained for Nm23-H1 and EDG2 and a statistically significant inverse correlation between these two proteins was revealed (r = -0.73; P = 0.004). The data indicate that Nm23-H1 down-regulation of EDG2 is functionally important to suppression of tumor metastasis.

nm23-h1 was the first metastasis suppressor gene to be identified in humans, with early studies demonstrating its ability to inhibit the metastatic potential of breast carcinoma and melanoma cell lines. This report outlines recent findings from our laboratory indicating that the metastasis suppressor function of NM23-H1 in human melanoma involves a spectrum of molecular mechanisms. Analysis of NM23-H1-dependent profiles of gene expression in human melanoma cell lines has identified a host of target genes that appear to mediate suppression of directional motility. Of particular interest is a subset of motility-suppressing genes whose regulation by NM23-H1 is independent of its known kinase and 3'-5' exonuclease activities. In parallel, we have recently observed that NM23-H1 expression appears to be required for genomic stability and for optimal repair of DNA damage produced by ultraviolet radiation and other agents. Thus, NM23-H1 might oppose not only the motile and invasive characteristics of metastatic cells but also the acquisition of mutations that drive malignant progression to the metastatic phenotype itself.

BACKGROUND: Alcohol consumption is an established risk factor for breast cancer metastasis. Yet, the mechanism by which alcohol promotes breast cancer metastases is unknown. The ability of cancer cells to invade through tissue barriers (such as basement membrane and interstitial stroma) is an essential step towards establishing cancer metastasis. In the present study, we identify and examine the roles of two genes, Nm23 and ITGA5, in alcohol-induced breast cancer cell invasion. METHODS: Human breast cancer T47D cells were treated with ethanol at various concentrations. Boyden chamber invasion assays were used to measure cellular invasive ability. The mRNA expression level of metastasis suppressor genes including Nm23 was determined by qRT-PCR. ITGA5 was identified using a qRT-PCR array of 84 genes important for cell-cell and cell-extracellular matrix interactions. Nm23 overexpression in addition to Nm23- and ITGA5 knock-down were used to determine the role of the Nm23-ITGA5 pathway on cellular invasive ability of T47D cells. Protein expression levels were verified by Western blot. RESULTS: Alcohol increased the invasive ability of human breast cancer T47D cells in a dose-dependent manner through the suppression of the Nm23 metastatic suppressor gene. In turn, Nm23 down-regulation increased expression of fibronectin receptor subunit ITGA5, which subsequently led to increased cellular invasion. Moreover, Nm23 overexpression was effective in suppressing the effects of alcohol on cell invasion. In addition, we show that the effects of alcohol on invasion were also inhibited by knock-down of ITGA5. CONCLUSIONS: Our results suggest that the Nm23-ITGA5 pathway plays a critical role in alcohol-induced breast cancer cell invasion. Thus, regulation of this pathway may potentially be used to prevent the establishment of alcohol-promoted metastases in human breast cancers.

OBJECTIVE: This study was designed to analyze expression patterns of estrogen receptor (ER), human epidermal growth factor receptor-2 (HER2/ERBB2), and nonmetastatic protein 23 (NM23-H1/NME1) proteins in patients with invasive ductal carcinoma and different menopausal status to identify their relationships with axillary lymph node metastasis. MATERIALS AND METHODS: 213 pre-menopausal and 177 post-menopausal women diagnosed with invasive ductal carcinoma were evaluated for ER, HER2, and NM23-H1 protein expression by immunohistochemistry. When HER2 immunoreactivity was equivocal (category 2+), specimens were confirmed by fluorescence in situ hybridization. RESULTS: ER expression showed no correlation with menopausal status or lymph node metastasis (each p > 0.05). However, expression of ER was associated with negative expression of HER2 (r = -0.214, p < 0.05) and positive expression of NM23-H1 (r = 0.137, p < 0.05) in the pre-menopausal group. Over-expression of HER2 was correlated with menopausal status (r = -0.107, p < 0.05) and lymph node metastasis in the ER-negative post-menopausal group (r = 0.222, p < 0.05). NM23-H1 was associated with less lymph node metastasis in the ER-positive pre-menopausal group (r = -0.237, p < 0.05). CONCLUSION: Our results indicated that expression patterns of ER, NM23-H1, and HER2 in primary breast cancer lesions warn that cells might have metastatic potential, which could assist clinicians to provide a more accurate prognosis and tailor therapeutic management for individual patients.

Breast cancer is a common cancer in women, with a highly variable course, from inoffensive to lethal. To find a more effective strategy for its treatment, sodium valproate has been tested as an anti-cancer drug; it is the only clinically available histone deacetylase inhibitor. However, data about the effects of sodium valproate on breast cancer are insufficient in both animals and humans; studies have yielded conflicting conclusions. In particular, little is known about the association between expression of the metastasis suppressor Nm23H1 gene and breast cancer. We hypothesized that sodium valproate regulates NM23H1 expression, and affects migration and/or invasion. We found that sodium valproate at concentrations of 0.8-3.2 mM inhibits migration and modulates Nm23H1 gene expression in a concentration-dependent manner. Confluent MDA-MB-231 cells were scratched by a micropipette tip after VPA treatment for 24 h; 24 h later, the scratch was almostly closed in the 0 mM VPA-treated cells, while the 3.2 mM VPA-treated cells migrated the slowest. The cell migration ratio exposed to 0.8, 1.6 and 3.2 mM VPA was about 66.67, 30.67 and 26.67% (P < 0.05). We also found evidence that sodium valproate upregulates NM23H1 expression, which is a clue to its anti-cancer mode of action. The NM23H1 gene expression was relative fold increased determined by Western blotting at 3.2 mM VPA. Collectively, these observations indicate that sodium valproate has potential for use in breast cancer treatment.

metastasis suppressor genes - unlike tumor suppressor genes - are defined by their capacity to control metastatic dissemination in vivo without affecting growth of the primary tumor. The first of these metastasis suppressor genes, NM23, was identified in 1988. Since then, expression of NM23 has been studied widely in human tumor cohorts, often with contradictory results. Not only is NM23 overexpressed in most human solid tumors when compared to healthy tissues, but also low expression of NM23 correlates with metastasis and poor clinical prognosis in the advanced stages of a number of epithelial cancer types, including melanoma, breast, colon, and liver carcinoma. This does not hold true, however, for other cancer types such as neuroblastoma and hematological malignancies, in which high NM23 expression correlates with more aggressive disease. Genetic alterations in the NM23 gene - loss of heterozygosity, spontaneous mutations and polymorphisms - are rarely found in tumors; thus, the metastatic potential of tumor cells is probably affected by NM23 protein levels. Three lines of evidence demonstrate the anti-metastatic activity of NM23: first, overexpression of NM23 in metastatic cell lines reduces their metastatic potential in xenograft models; second, the incidence of lung metastases is elevated in NM23 knockout mice prone to develop hepatocellular carcinoma, and, third, silencing NM23 by RNA interference confers a "metastatic phenotype" on non-invasive human epithelial liver and colon cancer cell lines. It appears that NM23 is crucial for inhibiting invasive migration, so acting at early stages of metastatic dissemination. The mechanistic basis of the metastasis suppressor function of NM23 and its regulated expression still remains obscure, however. Reactivation of expression of the endogenous NM23 gene in tumor cells, or stimulation of the pathways it controls, constitutes a promising avenue for anti-metastatic therapy.

AIM: to examine the presence of metastasis marker in various histological malignancies of ductal breast carcinoma using NM23HI protein. METHODS: paraffin blocks were obtained from 97 patients with invasive breast ductal cancer with 1, 2, 3 grade of malignancy from 2000 to early 2006 in several hospitals in Jakarta and Bandung. Examination began with histophatologic examination of eosin hematoxylin slides to diagnose the case of invasive ductal cancer and to get the data on the degree of histologic malignancy, metastasis or non-metastasis cases. It then continued to immunohistochemistry examination of NM23HI, and cytokeratin. RESULTS: subjects were 29-75 years old with the mean of 48.19 years; most subjects were 40-45 years old with malignancy grade 1 numbering 18.56%; grade 2, 45.36%; and grade 3, 36.1%. Ninety seven paraffin blocks were examined from 2000 to 2006. There was a significant relationship between NM23HI expression in primary tumor with the possibility of inhibition of invasion and metastasis 11 times of those of negative expression of NM23HI. The ROC curve showed that NM23HI expression was strongly correlated (r=0.816) sensitive and specific as metastasis marker. NM23HI expression did not show significant relationship with histologic degree of invasive ductal carcinoma. CONCLUSION: NM23HI expressions can be used as invasion and metastasis markers, but cannot be used as markers for the degree of histologic malignancy of invasive ductal cancer.

The nm23 gene has been proposed as a candidate metastasis-suppressor gene. In this study we used immunohistochemistry stain for polyclone antibody nm23 in 62 cases of buccal carcinoma. The results showed that both the epithelium of mucosa and the duct epithelium of minor salivary gland nearby the tumor were strong positive. The stains on tumor cells were various. 31.0% (9/29) of the cases with lymph-node metastasis were positive while 57.6% (19/33) of those with no lymph-node metastasis positive (P = 0.044). Histologically, 59.4% (19/32) of the cases were positive in Grade I, while 37.5% (6/16) positive in Grade II and 21.4% (3/14) in Grade III (P = 0.048). The positive rate increased with the patients survival time from less than 3 years to 3-5 years and more than 5 years correspondingly, but there was no statistical significance (P = 0.126). The results suggested that the expression of nm23 might be one of the factors influencing the lymph-node metastasis and prognosis of buccal cancer.

Epstein-Barr virus (EBV) is an oncogenic virus associated with a number of human malignancies including Burkitt lymphoma, nasopharyngeal carcinoma, lymphoproliferative disease and, though still debated, breast carcinoma. A subset of latent EBV antigens is required for mediating immortalization of primary B-lymphocytes. Here we demonstrate that the carboxy-terminal region of the essential latent antigen, EBNA-3C, interacts specifically with the human metastatic suppressor protein Nm23-H1. Moreover, EBNA-3C reverses the ability of Nm23-H1 to suppress the migration of Burkitt lymphoma cells and breast carcinoma cells. We propose that EBNA-3C contributes to EBV-associated human cancers by targeting and altering the role of the metastasis suppressor Nm23-H1.

In this study the authors detected both nm23-H1 and nm23-H2 mRNA levels in 52 tissues samples of patients with carcinoma of buccal mucosa (CBM) by Northern blotting method. The expression levels of both nm23-H1 and nm23-H2 mRNA in CBM were higher than those in normal buccal mucosa, leukoplakia, adjacent nontumorous mucosa, and lymph nodes with or without metastasis. The nm23-H1 mRNA expression levels in CBM with lymph nodes metastases were lower than those in CBM without metastasis, and were lower in metastatic lympho nodes (P < 0.05). Of the 11 cases with metastases, 9 cases (81.8%) showed low expression of nm23-H1 mRNA, of 19 cases without metastasis, 15 cases (78.9%) were high expression. No significance of nm23-H2 mRNA expression levels were found between CBM with and CBM without metastasis (P > 0.05). The expression of nm23-H1 and nm23-H2 mRNA showed no significant correlation with each clinical-pathological parameter examed. The result suggested that the expression of nm23-H1 mRNA significantly correlated inversely with lymph node metastasis in CBM, while the expression of nm23-H2 mRNA not. nm23 mRNA expression levels can provide a valuable predictor of lymph node metastases in CBM.

The nm23 gene is a conspicuous metastasis-suppressor gene. The authors detected both nm23-H1 and nm23-H2 mRNA levels in 47 tissues samples of patients with carcinoma of buccal mucosa (CBM) by quantitative reverse transcription PCR amplification. The results showed that expression levels of both nm23-H1 and nm23-H2 mRNA varied in normal buccal mucosa, leukoplakia, adjacent nontumorous mucosa, submandibular gland, CBM and lymph nodes with or without metastasis. The nm23-H1 mRNA expression levels in CBM with lymph nodes metastases were lower than those in CBM without metastases (P < 0.05), while no significance of nm23-H2 mRNA expression levels was found between CBM with and CBM without metastasis (P > 0.05). The results were comparative to those of Northern blotting of the same cases. The authors concluded that, as also in the study of Northern blotting, the expression of nm23-H1 mRNA significantly correlated inversely with lymph node metastasis in CBM, while the expression of nm23-H2 mRNA not. Q-RT-PCR was a useful method to detect the mRNA levels of nm23 gene in buccal carcinoma.

BACKGROUND AND OBJECTIVE: Previous studies have shown that nm23-H1 is a tumor metastasis suppressor gene. Nucleotide diphosphate kinase 1 (NDPK1) encoded by nm23-H1 is involved in cancer cellular differentiation, proliferation, apoptosis and metastasis. This study was to investigate the effects of nm23-H1 on proliferation and invasion of cervical cancer cells. METHODS: The eukaryotic expression vector pcDNA3.1-nm23-H1 was transfected into cervical cancer cells. Cell invasion potential was determined by the Transwell assay. Cell proliferation was measured by MTT assay and changes in cell cycle distribution were analyzed by flow cytometry (FCM). RESULTS: Compared with parent cells (Caski and SiHa) and vector control cells (Caski-3.1 and SiHa-3.1), the proliferation and invasion of pcDNA3.1-nm23-H1 transfected cells (SiHa-N and Caski-N) were apparently decreased (P<0.05); the proportions of G2/M and S cells were obviously decreased while that of G0/G1 cells was increased (P<0.05). However, transfection of nm23-H1 gene had no influence on proliferation, cell cycle and invasion of HeLa cells (P>0.05). CONCLUSION: nm23-H1 gene could inhibit proliferation and invasion of cervical cancer cells in a cell-dependent manner.

The product of the nm23 gene has been proposed as a candidate tumour metastasis suppressor protein. A strong association has been observed between reduced expression of the nm23 gene and acquisition of metastatic behaviour in some tumour cells, including breast cancer and melanoma, but not in others, such as neuroblastoma and colon, cervical and thyroid cancers. During the early gestation period both human and murine trophoblast cells exhibit in vitro invasive properties similar to those of neoplastic cells. Such invasive properties, however, disappear in the late stage of gestation. In the present study, we examined the abundance of nm23 mRNA from various fetal-maternal interface tissues (uterus, decidua, placenta and embryo) during early (day 8), mid (day 14) and late (day 18) stages of gestation in CD1 mice, in order to determine whether nm23 plays any anti-invasive and/or biological roles during gestation. nm23 was found to be expressed in all the tissues during the early and mid stages of gestation. The expression levels were, however, variable among different tissues and development stages. In the early stage, nm23 mRNA levels were the highest and similar among tissues from the uterus, decidua, placenta and embryo. In the mid stage, the mRNA levels were reduced significantly in the uterus, decidua and placenta, but not in the embryo. In the late stage, nm23 mRNA was further reduced to the extent that it could not be seen in the decidua, was barely seen in the uterus and was weakly present in the placenta. However, the mRNA level of the embryo in the late stage was still high and similar to the early stage. We also examined nm23 expression in trophoblast cells from normal human term placenta and a highly metastatic human choriocarcinoma cell line, JAR. nm23 expression was significantly higher in JAR than in normal placenta, indicating that nm23 does not appear to have an anti-metastatic function in this cell line. Several cytokines--interleukin 2 (IL-2), tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)--and prostaglandin E2 (PGE2) known to modulate tumour growth and metastasis were examined to determine whether they regulate nm23 expression in JAR in vitro. The B16F10 melanoma cell line was used as control. No effect was found in the JAR cell line, whereas TNF-alpha, IFN-gamma and PGE2 down-regulated nm23 expression in the B16F10 cell line.(ABSTRACT TRUNCATED AT 400 WORDS)FAU - Shi, Y.

Allelic deletions in the nm23, a metastasis suppressor gene, are known to occur in neuroblastomas, breast and colorectal carcinomas. Down-regulation of nm23 expression has been reported in various rodent and human tumor cells with high metastasis phenotype. Colorectal tumors showed overexpression of nm23. To elucidate the regulatory mechanisms of nm23, we isolated, cloned and sequenced the presumptive regulatory DNA fragment spanning the 5' region of the human nm23-H1 gene. The region's nucleotide sequence shows the presence of motifs typical for transcriptional elements such as TFIID, AP-1 and CTF/NF1. A common transcription initiation site is located at -136 upstream from the first ATG codon in placenta tissue, in breast, colorectal, prostate tumor cell lines and in primary colorectal tumor. Multiple transcription start sites were identified in tumor cell lines and colorectal tumor. When the promoter element was linked to a reporter gene, chloramphenicol acetyltransferase (CAT) and transfected in human 2fTGH cells, strong CAT activity was detected, which also showed that the presence of AP-1 and CTF/NF1 elements are essential for promoter activity. A detailed study of the structure and function of the promoter element of the nm23-H1 gene will help in understanding the regulatory mechanisms of nm23 expression and its role in tumor progression, especially in metastasis.

Nm23 is a putative metastasis suppressor gene and alterations in this gene have been reported in colorectal carcinomas suggestive of a role for nm23 in the dissemination of these tumours. In this study we used an affinity purified polyclonal antibody, ab-11, on formalin-fixed, paraffin-embedded sections of colorectal carcinomas from 46 patients in a three-stage avidin-biotin complex immunoperoxidase technique. Follow-up of these patients was until time of death or for 5 years, with a mean time of 31.2 months. Two observers scored the staining results from 1 to 3 according to the proportion of tumour cells positive. The association of nm23 staining with survival, sex, age, vascular invasion, and Dukes' stage was determined using Cox's regression model. The association of death from colorectal cancer and nm23 status reached marginal significance in this study (P = 0.0417). Moreover, there is some suggestion of a protective effect from nm23 as the relative risk of dying from colorectal cancer for each increment of nm23 positivity is 0.573 (95 per cent confidence limits 0.34-0.98).

BACKGROUND: The murine nm23 gene suppresses the metastatic behavior of malignant rodent tumor lines, and reduced nm23 expression correlates with increased likelihood of lymph node metastases in human breast cancers. More recent data have demonstrated the existence of two human nm23 gene homologues, nm23-H1 and nm23-H2, and have shown that deletion of nm23-H1 alleles occurs in some colon carcinomas associated with poor prognosis. These findings suggest that nm23-H1 encodes for suppression of colon carcinoma metastasis. In contrast, we have previously reported that total nm23 messenger RNA (mRNA) expression is increased to similar levels in colon tumors of both high and low metastatic potential. PURPOSE: This study was designed to reconcile our previous findings with the recent report of nm23-H1 allelic deletion in human colon cancers associated with poor prognosis. Our purpose was to examine human colon cancers for inactivation of two candidate metastasis suppressor genes, nm23-H1 and nm23-H2, either by mutation or by loss of gene transcription. METHODS: We used ribonuclease protection assays to analyze human colon tumors for the level of nm23-H1 (43 samples) and nm23-H2 (41 samples) transcript (mRNA) expression and the presence of mutations that could inactivate potential suppressor function. RESULTS: We detected only wild-type nm23-H1 and nm23-H2 mRNA. expression of nm23-H1 mRNA increased in 33 of 41 colon tumors, and expression of nm23-H2 mRNA was elevated in 28 of 41 colon tumors relative to that in matched normal mucosa. Increases in these mRNA levels were similar in tumors of both low and high metastatic potential. CONCLUSIONS: These results suggest that, despite correlation of nm23-H1 allelic deletions with colon cancers associated with poor prognosis, nm23-H1 and nm23-H2 alleles do not directly mediate metastasis suppression in colon carcinoma. Our results leave unexplained the observation that nm23-H1 allelic deletion correlates with metastatic potential of colon carcinomas. IMPLICATIONS: These findings also contrast with the demonstration of nm23 metastasis suppressor activity in murine melanoma and with the correlation of loss of nm23 expression in breast cancer with poor prognosis. It may be that metastasis suppression by the nm23 gene is a tissue-specific phenomenon.

This study evaluated the added significance of nm23-H1 to that of E-cadherin in determining metastatic proclivity in primary sporadic colorectal carcinomas (SCRCs). A clinical cohort of 52 SCRCs was examined for the significance of nm23-H1 and E-cadherin mRNA levels and E-cadherin protein expression levels into the progression of colorectal tumor invasion, determined by their relevance compared with conventional biological markers. A more than twofold decreased expression of nm23-H1 mRNA was reported in 28/52 (54%) of the carcinomas and was positively associated with the presence of nodal metastases and Astler-Coller stages B1 and B2 in 29% and 35% of the SCRCs, respectively. Reduced expression of E-cadherin mRNA was reported in 38.5% of the carcinomas and was similarly associated with stages Astler-Coller B1 and B2 in 27% of the SCRCs. Decreased E-cadherin immunohistochemical expression (grades II and III) was observed in 67% of the samples. E-cadherin mRNA and protein expression were significantly related to each other. The nm23-H1 (+)/E-cadherin (+) coexpression profile was observed in 31% and was significantly related to the absence of lymph node metastases in 31% and stages Astler-Coller B1 and B2 in 29% of the carcinomas examined. Furthermore, the nm23-H1 (-)/E-cadherin (+) coexpression profile was coupled to decreased E-cadherin immunohistochemical protein detection (grade II) in 21% of the cases examined. These findings suggest that impairment of nm23-H1 expression is an early event into the progression of colorectal metastasis that precedes E-cadherin transcriptional silencing in the majority of SCRCs examined. Nm23-H1 may therefore play an important role in suppressing the early steps of metastasis in sporadic cases of colorectal carcinomas.

AIM: To investigate the pathogenic mechanism of colon cancer at the molecular level and to elucidate the relationship between intercellular adhesion molecule-1 (ICAM-1) and nm23H(1) genes and Chinese patients with colon cancer. METHODS: DNA was extracted from paraffin-embedded materials. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to analyze MSI and LOH. expression of ICAM-1 was detected by Envision immuno-histochemistry. Experimental results were analyzed with Leica-Qwin computer imaging techniques and SPSS software of statistics. RESULTS: ICAM-1 expression of lymphatic endothelium was negative in normal colon and positive in colon cancer respectively. The number of lymphatics positive for ICAM-1 was gradually increased with degree of cancer invasion (P<0.01). In the group with metastasis of colon cancer, the number of lymphatics positive for ICAM-1 in lymph nodes was more than that in the group with no metastasis (P<0.01). The frequency of MSI, LOH and nm23H(1) protein was 26.67%, 20.00% and 53.33% in colon cancer, respectively. In TNM staging, MSI (43.75%) and nm23H(1) protein (81.25%) in stages I+II were detected more easily than the corresponding indexes (MSI: 7.14%, P<0.05 and nm23H(1): 21.43%, P<0.01) in stages III+IV. By comparison, the frequency of LOH (35.71%) in stages III+IV was more than that of LOH (6.25%, P<0.05) in stages I+II. LOH exhibited a rising trend along with the Dukes staging. nm23H(1) protein in the group of tubular adenocarcinoma (60.00%) was higher expressed than that in the group of mucoid adenocarcinoma (20.00%) (P<0.01), and exhibited a rising trend with the differentiation degrees of tubular adenocarcinoma. nm23H(1) protein in MSI positive group was higher expressed (75%) than that in MSI negative group (45.45%, P<0.05). CONCLUSION: The expression of ICAM-1 in lymphatic vessels is beneficial to the judgement of the invasion and metastasis ability of colon cancer and the anti-tumor immunity function, and shows an important clinical significance in predicting lymphatic metastasis of colon cancer. MSI and LOH may separately control the development of sporadic colon cancer with different pathways. LOH mostly arises in the late period of sporadic colon cancer and endows a high aggressive and poor prognostic phenotype. By compassion, MSI may be an early period molecule marker for sporadic colon cancer, enhanced expression of nm23H(1) protein can effectively inhibit colon cancer metastasis and improve prognosis of sporadic colon cancer patients.

tumor suppressor genes that reduce metastatic potential have been described in a variety of different tumor types. One of the main tumor metastasis suppressor genes is nm-23, which is a nucleoside diphosphate kinase. Two isotypes, nm-23H1 and nm-23H2, have been cloned and map to chromosome 17q21.3. In a variety of tumors, including colon cancer and breast cancer, loss of expression of nm-23 is associated with lymph node metastasis. In other organ systems, however, this relationship is not seen. In head and neck squamous cell carcinomas (HNSCC), there have been conflicting results regarding the association between nm-23 protein expression and metastatic potential. To further explore the tumor metastasis suppressor function of nm-23 in HNSCC, we studied high-stage laryngeal carcinomas, tumors with and without cervical lymph node metastasis for nm-23 protein expression and loss of heterozygosity of the gene locus. Twenty-five cases were included (11 cases with and 14 cases without metastasis). Loss of heterozygosity for the nm-23 gene locus was seen in 7 of 22 (32%) informative tumors. Using immunohistochemistry, most tumors expressed nm-23, though decreased expression was seen in 10 of 25 (40%) cases. Only 2 tumors showed negative expression. We did not find a correlation between either protein expression or loss of heterozygosity with metastatic disease or any other adverse prognostic factors in this group of high-stage laryngeal squamous cell carcinomas. These data imply that nm-23 may be tumor suppressor gene involved in HNSCC but that it may not function as a tumor metastasis suppressor in high-stage laryngeal carcinoma.

CONTEXT: NM23, a metastasis suppressor gene, may be associated with prognosis in patients with colorectal carcinoma. OBJECTIVE: To analyze NM23 expression and its association with the presence of lymph node and liver metastases and survival in patients operated on for colorectal carcinoma. METHODS: One hundred thirty patients operated on for colorectal carcinoma were investigated. Tissue microarray blocks containing neoplastic tissue and tumor-adjacent non-neoplastic mucosa were obtained and analyzed by immunohistochemical staining using a monoclonal anti-NM23 antibody. Immunohistochemical expression was assessed using a semiquantitative scoring method, counting the percentage of stained cells. The results were compared regarding morphological and histological characteristics of the colorectal carcinoma, presence of lymph node and liver metastases, tumor staging, and patient survival. Statistical analysis was performed using the Mann-Whitney test, the Kruskal-Wallis test and Fisher's exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. RESULTS: NM23 expression was higher in colorectal carcinoma tissue than in adjacent non-neoplastic mucosa (P<0.0001). NM23 protein expression did not correlate with degree of cell differentiation (P = 0.57), vascular invasion (P = 0.85), lymphatic invasion (P = 0.41), perineural infiltration (P = 0.46), staging (P = 0.19), lymph node metastases (P = 0.08), or liver metastases (P = 0.59). Disease-free survival showed significant association (P = 0.01) with the intensity of NM23 protein immunohistochemical expression in colorectal carcinoma tissue, whereas overall survival showed no association with NM23 protein expression (P = 0.13). CONCLUSIONS: NM23 protein expression was higher in neoplastic colorectal carcinoma tissue than in adjacent non-neoplastic mucosa, showing no correlation with morphological aspects, presence of lymph node or liver metastases, colorectal carcinoma staging, or overall survival. Disease-free survival was higher in patients with increased NM23 expression.

The nm23 gene is a potential metastasis suppressor gene originally identified using a murine melanoma cell line. The expression of nm23-H1 protein was examined immunohistochemically in 50 eligible patients with esophageal squamous cell carcinoma (ESCC). The expression was not correlated with other prognostic factors including lymph node metastases; however, overall survival rates of nm23-H1-negative patients were significantly shorter than those of nm23-H1-positive patients (P < 0.05). Furthermore, reduced expression of nm23-H1 was associated with shorter overall survival in patients with involved lymph nodes (P < 0.01), but not in patients without involved lymph nodes. These data support the conclusion that reduced expression of nm23-H1 may be associated with poor prognosis of ESCC patients, suggesting the value of nm23-H1 expression as a prognostic marker for ESCC patients, especially ESCC patients with involved lymph nodes.

The study was undertaken to determine the immunoexpression of protein products of nm23 genes which are thought to be potential metastasis suppressor genes, in esophageal squamous cell carcinoma, and to analyze their relationship to selected clinicopathological features (age, sex, tumour size, depth of invasion, presence of lymph nodes and distant metastasis, pathologic tumor stage, degree of cancer differentiation and vascular/lymphatic invasion), as well as to the overall survival. Immunohistochemical staining with monoclonal antibody against nm23 using LSAB2/HRP method on formalin-fixed, paraffin-embedded sections obtained from 32 tumors was performed. Eight tumors (25%) showed positive nm23 immunoexpression. There were no statistically significant differences between nm23-positive and nm23-negative groups with respect to all clinicopathological features studied. The positive nm23 status was related to a worse prognosis but this was not significant. The results may suggest that nm23-status is not associated with metastatic ability and prognosis in esophageal squamous cell carcinoma, but such thesis requires further study on a larger population.

The esophageal squamous cell carcinoma (ESCC) has high incidence in Shaanxi Province of China. More and more researches indicated that human papillomavirus type 16 (HPV16) might play an important role in carcinogenesis of ESCC but the relationship between HPV16 and CD44v6, nm23H1 has not been elucidated. HPV16 was detected by amplifying HPV16 E6 gene through polymerase chain reaction (PCR) method and the expression of CD44v6, nm23H1 in 40 ESCCs and fifteen normal esophageal mucosa (NEM) from Shaanxi Province was examined by Streptavidin-Peroxidase (SP) method using monoclonal antibody specific to CD44v6 and nm23H1. The positive rates of HPV16 E6 gene, CD44v6 and nm23H1 were 60% (24/40), 65% (26/40) and 45% (18/40) respectively in ESCCs and 26.67% (4/15), 33.33% (5/15) and 86.67% (13/15) respectively in NEMs. There exited statistical difference for HPV16, CD44v6 and nm23H1 between NEMs and ESCCs respectively (p < 0.05). The relationship between HPV16 and the expression of CD44v6 in ESCCs was statistical significance (P = 0.021), but no significant correlation was found between HPV16 and the expression of nm23H1 (P = 0.436) in ESCCs. The infection rate of HPV16 had no statistical difference in all pathological features we observed, but the expression rates of CD44v6 and nm23H1 had statistical correlation with invasion (p = 0.001, 0.013) and lymph nodes metastasis (p = 0.014, 0.002) respectively. In different histology grade of ESSCs, the relationship between HPV16 and CD44v6 was statistical significance in grade I (p = 0.044) but was not in grade II (p = 0.165) and grade III (p = 0.658), however as to the expression of nm23H1 there exited no statistical significance in all histology grades of ESCC (p > 0.05). The expression rates of CD44v6 and nm23H1 were statistically different between grade I and II (p = 0.004, 0.016) respectively and between grade I and grade III (p = 0.014, 0.020), but not statistically different between grade II and III (p = 0.792, 0.943) respectively. Our data firstly suggested that there existed the statistical relationship between the infection of HPV16 and the expression of CD44v6 in ESCCs and that HPV16 may be involved in invasion and metastasis of ESCC.

beta-catenin has emerged as a key regulator of Wnt signaling pathway, which plays an important role in the development and progression of various cancers. Its accumulation in nucleus of the esophagus squamous epithelium might be the crucial step for the carcinogenesis of esophageal squamous cell carcinoma (ESCC). To detect the proteins correlated with beta-catenin function, we used the established cell lines of pGen-3-con (Eca109 cells transfected by control vector) and pGen-3-CTNNB1 (Eca109 cells transfected by beta-catenin siRNA) as cell models for further analysis. Two-dimensional gel electrophoresis technology was performed to separate the proteins of pGen-3-con and pGen-3-CTNNB1 cell lines, respectively. The differential protein spots were analyzed by software analysis, subjected to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Consequently, 13 differentially expressed proteins between the two cell lines were identified, of which 14-3-3sigma, prohibitin, and nm23-H1 were further verified by western blotting and quantitative real-time reverse transcriptase-polymerase chain reaction. Then, the tissue microarray and immunohistochemical analysis were employed to research their relationship in ESCC and their corresponding normal mucosa tissues. The upregulation of prohibitin or the downregulation of 14-3-3sigma and nm23-H1 proteins was significantly associated with the proliferation, invasion depth, and lymph node metastasis of ESCC. There were statistically significant correlations between the expression of beta-catenin and the three proteins. The results presented here might provide potential protein markers to elucidate the mechanism of beta-catenin-mediated biologic characteristics for ESCC.

OBJECTIVE: To investigate the expression of two tumor metastasis suppressor genes nm23 and KAI1 in gallbladder adenocarcinoma, and their clinicopathological significance. METHODS: Specimens and clinical data from 31 gallbladder adenocarcinoma patients were collected. Histopathological grading and the expression of nm23 and KAI1 were detected by HE and immunohistochemical staining, respectively. All cases were followed up for at least three years. RESULTS: Immunohistochemical staining showed that the positive rate of nm23 and KAI1 proteins was 71.0% (22/31) and 61.3% (19/31), respectively. The positive expression rates of nm23 and KAI1 proteins in the early stage carcinomas were significantly higher than those in the moderate and advanced stage ones (P exact = 0.0051 and P exact = 0.0084), and both had an negative correlation with clinicopathologic stage (P trend = 0.0047 and P trend = 0.0058). There was a significant difference in the expression of nm23 and KAI1 proteins among well, moderately and poorly differentiated carcinomas (P exact = 0.0328 and P exact = 0.0020). The expression of nm23 and KAI1 was positively correlated with histopathological grade (P trend = 0.0086 and P trend = 0.0006). There was also a significant difference in the expression of nm23 and KAI1 proteins between 17 survival and 14 dead patients (P exact = 0.0038 and P exact = 0.0001 ). A synergistic effect of nm23 and KAI1 protein on the survival was observed , and seemed to be more important than any individual gene alone (P exact = 0.0005). CONCLUSION: The expressions of nm23 and KAI1 proteins are negatively correlated with clinical stage, but positively with histopathological grade in gallbladder adenocarcinoma. These two tumor metastasis suppressor genes may act synergistically to inhibit the tumor metastasis.

BACKGROUND & OBJECTIVE: Molecular biological studies indicated that abnormal expression of cyclooxygenase-2(COX-2), p53, proliferating cell nuclear antigen(PCNA), and nm23 correlate with the development of gastric cancer, but the relationship between abnormal expression of above gene proteins and the biological behavior of gastric cancer was not yet clear. This study was designed to investigate the relationship between expressions of COX-2, p53, PCNA, and nm23 and the clinicopathological behavior in gastric cancer. METHODS: The expressions of COX-2, p53, PCNA, and nm23 were detected by using immunohistochemical(SP) method. RESULTS: (1) The overexpression rate of COX-2 in gastric cancer was 70.4% (50/71), and the overexpression of COX-2 was correlated to tumor size, gross morphology, histological type, lymph node metastases, and clinicopathological stage of gastric cancer (P < 0.05, or P < 0.01). (2) The overexpression rates of p53 and PCNA in 71 surgically removed specimens from the patients with gastric cancer were 74.6% and 78.9%, respectively. And the expressions of p53 (86.7%) and PCNA (88.8%) in gastric cancer patients with lymph node metastasis were significantly higher than those in patients without lymph node metastasis (53.8% and 61.5%) (both, P < 0.05), respectively. Moreover, expressions of p53(52.2%) and PCNA(60.9%) in stage I + II patients were lower than those(85.4% and 87.5%) in stage III + IV (both, P < 0.05). (3) The low-expression of nm23 in gastric cancer was 71.8%, and its low-expression rate of nm23 in patients without lymph node metastasis(88.5%) and in stage I + II (91.3%) were much higher than those in the patients with lymph node metastasis (62.2%) and in stage III + IV (62.5%) (respectively, P < 0.05). (4) The differences of histological types, depth of invasion, lymph node metastasis and clinical stage were significantly different(P < 0.05 or P < 0.001), between the patients with two or more genes expression of COX-2, p53, PCNA, and nm23 and those with less one gene expression. CONCLUSION: Our results suggest that abnormal expressions of COX-2, p53, PCNA, and nm23 associate with malignant potential, lymph node metastasis and clinical stage, and they might therefore play a role in development of gastric cancer.

AIM/BACKGROUND: nm23 is suggested to represent a new class of metastasis suppressor genes. An inverse correlation between nm23 expression level and metastatic potential has been demonstrated in different malignancies. This study evaluated the prognostic value of nm23 in gastrointestinal stromal tumors (GISTs). METHODS: Immunohistochemical expression level of nm23 was studied in a total of 32 patients with localized GISTs. The relationship between the expression level of nm23 and patient outcome was investigated. RESULTS: A tumor size of 10 cm or more and a mitotic rate of 10 or more per 50 high-power fields were not significantly associated with the metastasis risk (p = 0.60 and 0.55, respectively). tumors with high expression of nm23 tended to have significantly lower metastatic potential (p = 0.02). The median survival was significantly longer in patients with high expression of nm23 (p = 0.007). CONCLUSION: These results suggest that expression level of nm23 may be considered as a prognostic predictor in GISTs. Future studies with larger patient numbers will be essential to confirm the prognostic significance of nm23 in patients with GIST.

AIM: To explore the expression of heparanase gene, CD44v6, MMP-7 and nm23 proteins in human gastric carcinoma as well as their relationship with the clinicopathological factors. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was used to measure the expressions of heparanase mRNA in 43 human gastric carcinomas and 10 adjacent normal gastric tissues. Streptavidin-peroxidase (S-P) two step method was used to measure the immunohistochemical expression of CD44v6, MMP-7 and nm23 protein in 43 human gastric carcinomas. RESULTS: The expression of heparanase gene was positive in 29 cases of gastric cancer with a positive rate of 67.4%, which was significantly higher than those in adjacent normal gastric tissues (P<0.05). The heparanase mRNA expression was significantly related to advanced stage of disease, serosal infiltration, lymph node metastasis and size of tumors (P<0.05), but not related to tumor location, gross and histological types of the cancer, peritoneal dissemination and liver metastasis (P>0.05). The positive rate of CD44v6, nm23 and MMP-7 proteins was 76.7%, 37.2% and 60.5%, respectively. The CD44v6 protein expression was significantly related to serosal infiltration, lymph node metastasis and TNM stage of disease (P<0.05). The nm23 protein expression was significantly related to the tumor gross appearance, lymph node and peritoneal metastasis (P<0.05). The MMP-7 protein expression was significantly related to serosal infiltration and TNM stage of disease (P<0.05). In an attempt to measure the association between these agents, we found that the expression of heparanase mRNA had significantly negative correlation with the expression of CD44v6 and MMP-7 protein (P<0.05), the expression of MMP-7 protein had significantly positive correlation with the expression of CD44v6 protein (r=0.568, P<0.05), the expression of MMP-7 protein had no correlation with the expression of nm23 protein (P>0.05), and the expression of nm23 protein had no correlation with the expression of CD44v6 protein (P>0.05). CONCLUSION: Despite their different mechanisms of action, heparanase, CD44v6 and nm23 may play important roles in the invasive infiltration and lymph node metastasis in gastric carcinomas. Instead of metastatic spreading, MMP-7 may be just related to the invasion of gastric carcinomas. However, co-detection of these factors may be important to predict metastatic spreading in such patients.

To investigate microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D17S396, D17S579 and D17S855, and their effect on the expression of nm23H1 and BRCA1 of gastric cancer, which would provide experimental basis for clinical treatment and prognosis analysis of gastric cancer. DNA was extracted from paraffin-embedded materials. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to analyze MSI and LOH. expression of nm23H, and BRCA1 was detected by Envision immuno-histochemistry and Leica-Qwin computer imaging techniques. In the forty cases of gastric cancer, the frequency of MSI, LOH and nm23H1 protein were 20.00%, 17.50% and 55.00% respectively at locus D17S396, while at locus D17S579, the frequency of MSI, LOH and BRCA1 protein were 22.50%, 15.00% and 37.50% respectively; at locus D17S855, the frequency of MSI, LOH and BRCA1 of thirty-seven cases were 18.92%, 18.92%, 37.84% respectively. In tumor node metastasis (TNM) staging, at locus D17S396, D17S579 and D17S855, MSI in stages I + II appeared more frequently than that in stages III + IV, while LOH appeared the contrary tendency. In the group of metastasis of gastric cancer, MSI had a less frequency (5.00%) than that with no metastasis (35.00%, P < 0.05) at locus D17S396, but LOH appeared more frequently (30.00%) than that with no metastasis (5.00%, P < 0.05). At locus D17S579, MSI had an increasing tendency with the degree of tumor differentiation (50.00% in high differentiation cases, 20.00% in middle differentiation cases, and 0% in low differentiation cases, P < 0.05). The frequency of nm23H1 and BRCA1 protein in stages TNM I + II was higher than that in stages TNM III + IV; and that in higher differentiation cases was higher than in poor differentiation cases. The frequency of nm23H1 protein in the group of metastasis (30.00%) was less than that with no metastasis significantly (80.00%, P<0.01). The frequency of nm23H1 protein in the group positive to MSI (87.50%) was higher than that in the group negative to MSI (46.88%, P < 0.05). However, nm23H1 protein in group positive to LOH (14.29%) was lower than that in the group negative to LOH (63.64%, P < 0.05). The frequency of BRCA1 protein in the group positive to MSI (66.67%) was more than that in the group negative to MSI (29.03%, P < 0.05). The results of experiments indicate that MSI and LOH may separately control the development of sporadic colon cancer with different pathways. MSI may be an early period molecule marker for sporadic colon cancer, enhanced expression of nm23H1 protein can effectively inhibit colon cancer metastasis and improve prognosis of sporadic colon cancer patients. By comparison, LOH mostly arises in the late period of sporadic colon cancer and endows a high aggressive and poor prognostic phenotype. nm23H1 protein could effectively restrain gastric cancer metastasis and development; and BRCA1 protein could restain tumor from becoming lower differentiation.

The correlations between the expression of nm23H1 mRNA,VEGF-C mRNA, VEGFR-3 and neoangiogenesis, hyperplasia of micro-lymphatic, tumor metastases in gastric cancer were studied, the results supplied an experimental foundation for clinical treatment and prognosis. All the specimens were marked by VEGFR-3 antibody, the expressions of nm23H1 mRNA, VEGF-C mRNA were detected in 78 cases of gastric cancer in situ hybridization with EnVision and Leica-Qwin computer image analysis system. The Weidners highest vessel density counting method was used to analyse micro-lymphatics count (MLC) of the specimens, and the patients viability after operation was investigated. The positive expression of nm23H1 mRNA in gastric cancer was 69.23% (54 cases). There was negative correlation between the positive expression of nm23H1 mRNA and lymph node metastasizing, TNM staging, MLC (P < 0.01 or P < 0.05). The expression of VEGF-C mRNA was 46.15% (36 cases). It was positive related to the lymph node metastasis, TNM stages, MLC in gastric cancer (P < 0.01 or P < 0.05), and was significantly different compared with adjacent nontumorous tissue (P < 0.01 or 0.05). The expression of nm23H1 in stage I and II gastric cancer was high, while in stage III and IV the expression was lower or even none. The MLC (8.37 +/- 2.29/mm2) in gastric cancer was higher than that in adjacent nontumorous tissue (4.82 +/- 3.48/mm2), which has statistical significance (P < 0.05). There was negative correlation between the expression of nm23H1 mRNA and VEGFR-3 (p < 0.05, r = 0.8479), but positive correlation between VEGF-C mRNA and VEGFR-3 (P < 0.05, r = 0.8362). The MLC (10.82 +/- 2.51/mm2) in those who died in five years (48 cases) was higher than that (6.53 +/- 2.09/mm2) in those who were still alive, the difference has statistical significance (P < 0.05). In different tumor pathology grading, different differentiation, the nm23H1 positive expression is significantly different (P < 0.05). Higher the nm23H1 expressed, lower the tumor lymph metastasized, but the viability rate was higher, so the nm23H1 gene was thought to have the effect of suppressing gastric cancer occurring and lymph metastasizing. Higher the VEGF-C mRNA expressed, higher the tumor lymph metastasized, but the viability rate was lower. There is close correlation between the VEGFR-3 expression and gastric cancer lymph metastasizing. The higher MLC level indicated neoangiogenesis in gastric cancer. VEGF-C promote neoangiogenesis induced by tumor and play an important role in lymph metastasizing.

PURPOSE: metastasis remains an incurable common complication in patients with gastric cancer. A variety of theories have been proposed to explain the inefficiency of the metastatic process. To compare protein expression of metastasis-related genes (nm23, KISS1, KAI1 and p53) between primary tumours and metastatic tumours may be useful in illustrating these theories. METHODS: metastasis-related tissue microarrays (including normal tissues, primary tumours, nodal metastases and liver metastases) were constructed. The protein expression of nm23, KISS1, KAI1 and p53 in lymph node and liver metastases from advanced gastric cancer specimens was mainly examined by immunohistochemical staining in relation to primary tumours. RESULTS: Immunohistochemical staining showed reduced protein expression of nm23, KISS1 and KAI1 in lymph node and liver metastases compared with primary tumours. Results for p53 were to the contrary. CONCLUSIONS: Our investigations revealed a tendency of reduced protein expression of metastasis suppressor genes nm23, KISS1 and KAI1 in gastric cancer with the progress of metastasis. This means that the progression theory is an important determinant of metastatic efficiency.

BACKGROUND: The expression of the nm23 gene has been associated with the development of metastasis. Numerous studies have shown down-regulation of nm23 expression in metastatic breast and colon cancer. The expression of the putative metastasis-suppressor gene nm23 in gastric carcinoma is controversial. The aim of this study was the analysis of nm23 expression in a large series of gastric cancer patients. PATIENTS AND METHODS: In a retrospective immunohistochemical study specimens obtained from 116 gastric cancer patients (mean age 64 years; range: 33-85) who had undergone gastrectomy with extended lymphadenectomy were analyzed. Nm23 expression in the tumor epithelium was studied by immunohistochemistry followed by a semi-quantitative (score 0-3) evaluation. Statistical analysis including Chi-square test, uni- and multivariate survival analyses were performed. RESULTS: The nm23 staining pattern was positive (score 2-3) in 100 (86.2%) specimens and negative (score 0-1) in 16 (13.8%) samples. Lymph node metastasis was found in 65% of the patients. No significant correlations could be determined between nm23 expression and other variables such as gender, age, tumor differentiation, WHO-, Lauren-, Goseki-, or Ming-classification. The intensity of nm23 staining in the tumor cells was not significantly correlated with depth of tumor infiltration (T-stage), lymph node metastasis (N-stage), distant metastasis (M-stage), UICC-stage, or prognosis. CONCLUSION: Our series did not show a correlation of nm23 expression in terms of lymph node and distant metastasis or prognosis in gastric cancer patients.

AIM: To investigate the changes of histology and expression of MMP-2 and nm23-H1 in primary and metastatic gastric cancer. METHODS: One hundred and seventy-seven gastric cancer patients with lymph node and/or distal metastasis between 1997 and 2001 were reviewed. Differences in histology of the primary and metastatic gastric cancer were assessed. MMP-2 and nm23-H1 immunoreactivity was compared in 44 patients with tumor infiltration to the serosa layer. RESULTS: Poorly and moderately differentiated metastatic gastric cancer was found in 88.7% (157/177) and primary gastric cancer in 75.7% (134/177) of the patients. The histological type of metastatic gastric cancer that was not completely in accordance with the preponderant histology of primary gastric cancer was observed in 25 patients (14.1%). MMP-2 immunoreactivity in metastatic gastric cancer was significantly stronger than that in primary gastric cancer, while nm23-H1 immunoreactivity showed no difference in primary and metastatic gastric cancer. CONCLUSION: metastatic gastric cancer presents more aggressive histological morphology and higher MMP-2 immunoreactivity than primary gastric cancer. This heterogeneity may elicit a possible mechanism of gastric cancer metastasis.

Single nucleotide polymorphisms (SNPs) in the promoter regions of non-metastatic cells 1 gene (NME1) may attribute to the changing of promoter activities. In addition, high NME1 protein levels are correlated with negative lymph node metastasis in gastric cancer. This study evaluated possible associations between SNPs in NME1 gene and gastric cancer susceptibility, clinicopathological parameters, or survival. We obtained formalin-fixed paraffin-embedded tissues from 404 gastric cancer patients and blood samples from 404 controls. SNPs were genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A significant correlation between SNPs and lymph node metastases risk was found. Patients carrying TT genotype in rs16949649, AA genotype in rs3760468, TT genotype in rs3760469, CC genotype in rs2302254, and GG genotype in rs34214448 were correlated to greater numbers of lymph node metastases (P = 0.023 in rs16949649; P = 0.015 in rs3760468; P = 0.043 in rs3760469; P = 0.008 in rs2302254; and P = 0.021 in rs34214448, respectively). Moreover, the haplotype TATCG were associated with positive lymph node metastasis (P = 0.039) and lymphovascular invasion (P = 0.048) compared to the haplotype CTGTT carriers. Furthermore, patients carrying AA genotype in rs3760468 or the haplotype TATCG had poor survival in T4 subgroup (P = 0.038 in univariate and P = 0.014 in multivariate analysis for rs3760468, and P = 0.017 in univariate and P = 0.012 in multivariate analysis for TATCG, respectively). In conclusion, SNPs in NME1 gene may play an important role in regulating lymph node metastasis and, thus, affect survival in T4 subgroup of gastric cancer in a Northern Chinese population.

The nm23, encoding a protein identical to nucleoside diphosphate (NDP) kinase, is thought to be a candidate metastasis suppressor gene. Low expression of nm23 at the protein or/and mRNA levels was associated with metastatic potential of several types of rodent and human tumors. expression and anti-metastatic action of nm23/NDP kinase gene products of human head and neck cancer (squamous cell carcinoma) cell lines were investigated. Among six cell lines examined, only those which showed metastatic potential exhibited reduced levels of nm23-H2 protein with no apparent change in nm23-H1 protein levels as compared with non-metastatic cells. When either nm23-H1 or nm23-H2 cDNA was transfected into a high metastatic cell line, only cell clones expressing higher levels of nm23-H2 protein above control levels displayed a reduced number of lung foci in an experimental metastasis assay. The data suggested that nm23-H2 gene product possesses the metastasis suppressor activity in human head and neck cancer cells.

OBJECTIVE: To study the metastasis feature of the primary and metastatic lymph node lesions in supraglottic or hypopharyngeal cancer. METHODS: The expression of CD44 and nm23-H1 in specimens from the primary and metastatic lymph node lesions of the 41 cases with supraglottic or hypopharyngeal cancer were studied with immunohistochemistry method and flow cytometry. RESULTS: No correlation was found between the expression of CD44, nm23-H1 and the tumor differentiation of the supraglottic or hypopharyngeal cancer, but their expression related with the clinical staging. The CD44 and nm23-H1 positive expression rates in the primary and metastatic lymph node lesions were 75.6% (31/41), 85.4% (35/41) and 34.1% (14/41), 26.8% (11/41) respectively (P >0.05). The average fluorescence index of CD44 and nm23-H1 in the primary and metastatic lymph node lesions were 1.27 +/- 0.18, 1.33 +/- 0.16 and 1.11 +/- 0.19, 1.08 +/- 0.15 (x +/- s) respectively (P >0.05). CONCLUSIONS: The expressions of CD44 and nm23-H1 in the metastatic lymph node tumor had no difference compared with that in primary tumor of the supraglottic or hypopharyngeal cancer. The difference of metastasis potentials between the primary and metastatic lymph node lesions in the same patient was not proved in this study and should be further investigated from multiple oncogens markers.

OBJECTIVES: To investigate the clinical significance of nm23-H1 gene product in sarcomatous cancer cells, because this is known as a tumor-metastasis suppressor. Renal cell carcinoma with sarcomatoid cancer cells is characterized by high malignant potential and a poor prognosis. METHODS: We investigated the expression of nm23-H1 gene product in the carcinomatous and sarcomatous component (CC and SC) of renal cell carcinoma using immunohistochemical techniques and the relationships between the expression and clinicopathologic features. We also examined the expression of matrix metalloproteinase (MMP)-2, MMP-9, sialyl Lewis X, and c-erbB-2 in the SC because these proteins are regulated by the nm23-H1 gene or its products. RESULTS: We examined 20 renal cell carcinoma specimens that contained an SC and CC. The CC of 12 of the 20 tumors stained positively for nm23-H1 gene product. In contrast, the SC of only 3 of the 20 stained positive. The reduced expression of nm23-H1 gene product in the SC correlated significantly with tumor invasion (P <0.01), but not with tumor size or metastasis. In contrast, the expression of the nm23-H1 gene product in the CC was not associated with these pathologic features. expression of the nm23-H1 gene product correlated negatively with MMP-2 expression (r = -0.48, P = 0.03). Other factors did not show such significant correlations with nm23-H1 gene product expression. CONCLUSIONS: Our results suggest that low expression of nm23-H1 gene products may play important roles in tumor invasion, and that this process is mediated in part by overexpression of MMP-2.

The nm23 gene is a metastasis suppressor gene. There are now two known isotypes of human nm23, namely nm23-H1 and nm23-H2. Several investigators have reported that reduced expression of nm23-H1 is associated with metastasis or disease progression in some of the tumor cases. In this study, the expression of nm23-H1 mRNA in the human laryngeal squamous cell carcinoma was investigated by Northern blot analysis. The levels of nm23-H1 mRNA were higher in cancers (n = 31) than in the adjacent normal tissues (n = 7), but the difference was not significant (P > 0.05). There was a significant decrease of nm23-H1 mRNA levels in tumors with lymph node metastasis (n = 11) as compared with those without (n = 20) (P < 0.01). cancer tissues in clinical stages III-IV. (n = 23) also exhibited lower levels of nm23-H1 mRNA expression than those in stages I-II (n = 8) (P < 0.05). These results suggest that the nm23-H1 gene may play a role in the suppression of lymph node metastasis or disease progression in laryngeal cancer.

tumor suppressor genes that reduce metastatic potential have been described in a variety of different tumor types. One of the main tumor metastasis suppressor genes is nm-23, which is a nucleoside diphosphate kinase. Two isotypes, nm-23H1 and nm-23H2, have been cloned and map to chromosome 17q21.3. In a variety of tumors, including colon cancer and breast cancer, loss of expression of nm-23 is associated with lymph node metastasis. In other organ systems, however, this relationship is not seen. In head and neck squamous cell carcinomas (HNSCC), there have been conflicting results regarding the association between nm-23 protein expression and metastatic potential. To further explore the tumor metastasis suppressor function of nm-23 in HNSCC, we studied high-stage laryngeal carcinomas, tumors with and without cervical lymph node metastasis for nm-23 protein expression and loss of heterozygosity of the gene locus. Twenty-five cases were included (11 cases with and 14 cases without metastasis). Loss of heterozygosity for the nm-23 gene locus was seen in 7 of 22 (32%) informative tumors. Using immunohistochemistry, most tumors expressed nm-23, though decreased expression was seen in 10 of 25 (40%) cases. Only 2 tumors showed negative expression. We did not find a correlation between either protein expression or loss of heterozygosity with metastatic disease or any other adverse prognostic factors in this group of high-stage laryngeal squamous cell carcinomas. These data imply that nm-23 may be tumor suppressor gene involved in HNSCC but that it may not function as a tumor metastasis suppressor in high-stage laryngeal carcinoma.

OBJECTIVE: To explore the relationship between the expressions of HER-2/neu, nm23 protein and local invasion and lymp node metastasis of LSCC. METHOD: The expression of HER-2/neu, nm23 protein was detected by immunohistochemical technique in 56 patients with human LSCC. RESULT: The positive expression rate of HER-2/neu protein in LSCC was not correlated with histological grade (P > 0.05). There was a significant relationship between the expression of HER-2/neu and the clinical stages, lymph node metastasis (P < 0.05, P < 0.01). The positive expression rate of nm23 protein was significantly higher in the group without lymph node metastasis than those with lymph node metastasis (P < 0.01). The negative correlation was found between the nm23 expression and lymph node metastasis of LSCC. Furthermore, the negative correlation was found between the positive expression of HER-2/neu protein and the positive expression of nm23 protein in the group with lymph node metastasis. CONCLUSION: The abnormal expression of HER-2/neu, nm23 protein play an important role in development and lymph node metastasis of LSCC.

OBJECTIVE: To study the expression of CD44 and nm23-H1 gene proteins and their clinical significance in laryngeal carcinoma. METHOD: The expression of CD44 and nm23-H1 proteins were detected by immunohistochemistry method in 40 cases with laryngeal carcinoma, 20 adjacent carcinoma tissues and 12 cases normal laryngeal mucosa tissues. RESULT: The expression of CD44 and nm23-H1 proteins in laryngeal carcinoma were much higher than that in normal laryngeal mucosa. The expression of CD44 protein in laryngeal carcinoma with metastatic lymph node was higher than that in laryngeal carcinoma without metastatic lymph node, but nm23-H1 protein lower. The expression of CD44 protein was positively correlated with the metastasis, clinical staging and pathological classification but not correlated with T classification of laryngeal carcinoma. The expression of nm23-H1 protein was negative correlation with the metastasis and clinical staging of laryngeal carcinoma. CONCLUSION: CD44 and nm23-H1 gene proteins play an important coordinated regulation role in the carcinogenesis, development and metastasis of laryngeal carcinoma and will probably become the key biological marks in the judging and evaluating prognosis of laryngeal carcinoma.

For the purpose of investigating the relationship between the metastatic potential of the tumor as well as the expression of nm23-H1 mRNA, and for determing the location of the positive sites in the cells, tumor metastasis suppressor gene nm23-H1 in human hepatocellular carcinoma (and the nonneoplastic area surrounding the tumor) was detected by in situ hybridization using digoxiginin-labeled nm23-H1 antisense complementary RNA probe. The primary results indicated (i) positive results of in situ hybridization are presence of granules or masses in the cytoplasm; (ii) the less expression of nm23-H1 mRNA, the higher metastatic rate of the human hepatocellular carcinoma (P < 0.05); (iii) expression of nm23-H1 mRNA dose not correlate with some other factors such as tumor size and the background of other liver diseases.

AIMS/BACKGROUND: Decreased expression of nm23, a putative metastasis suppressor gene, has been reported to be related to either intrahepatic metastasis or a poor prognosis in hepatocellular carcinoma (HCC). The aim of this study was to elucidate the true role of nm23-H1 expression in both intrahepatic and distant metastases of HCC. METHODS: Thirteen patients with single-nodule HCC, seven patients with HCC having satellite nodules and seven patients with HCCs having extrahepatic metastases were included in this study. The expression of nm23-H1 protein was immunohistochemically examined in both primary and metastatic nodules. RESULTS: Ten of 13 single-nodule HCCs were found to overexpress nm23-H1 protein. All main tumors, having satellite nodules, were found to overexpress nm23-H protein, except for two HCCs, which only partially expressed nm23-H1 protein. Regarding the nm23-H1 expression in intrahepatic metastases, most nodules overexpressed the protein. The expression of nm23-H1 was found to be low in only one intrahepatic metastasis specimen, while its primary tumor was also found to show a low expression of nm23-H1 protein. Microscopic portal vein invasion was found in three of the five patients studied, and all cancer cells in portal invasion overexpressed nm23-H1 protein. Nm23-H1 protein was expressed in all distant metastatic tumors and the staining intensity of most metastatic nodules was similar to that of the primary tumors. CONCLUSIONS: Our study demonstrated that nm23-H1 expression did not always decrease but instead tended to increase at both intrahepatic and extrahepatic metastatic sites. Based on these findings, nm23-H1 expression is not considered to be a reliable indicator of either intrahepatic or distant metastasis in HCC.

One of the most aggressive cancers is hepatocellular carcinoma, which is associated with a very poor patient outcome due to a high recurrence rate and metastatic spread. NM23, the first metastasis suppressor gene to be identified, has been widely studied in human cancers. However, conflicting results have been obtained depending on the tumor type and the evaluation protocol. The current knowledge of NM23 as a diagnostic and/or prognostic marker in hepatocellular carcinoma is reviewed herein. Most studies demonstrate an inverse association between the expression of NM23-H1 and the metastatic potential, which is not observed with the closely related NM23-H2 isoform. Transfection of metastatic hepatoma cells with NM23 reduced their metastatic potential, as for other tumor cell lines. The demonstration of a causative role of NM23 in metastatic dissemination in a mouse model of hepatocarcinoma suggests that hepatocarcinoma-derived cells could be good models for the analysis of the molecular mechanisms involved in NM23 action.

Nm23-H1, also known as NDPK-A, was the first of a class of metastasis suppressor genes to be identified. Overexpression of Nm23-H1 in metastatic cell lines (melanoma, breast carcinoma, prostate, colon, hepatocellular, and oral squamous cell carcinoma) reduced cell motility in in vitro assays and metastatic potential in xenograft models, without a significant effect on primary tumor size. The mechanism of Nm23-H1 suppression of metastasis, however, is incompletely understood. Nm23-H1 has been reported to bind proteins, including those in small G-protein complexes, transcriptional complexes, the Map kinase, the TGF-beta signaling pathways and the cytoskeleton. Evidence supporting these associations is presented together with evidence of resultant biochemical and phenotypic consequences of association. Cumulatively, the data suggest that part of the anti-metastatic function of Nm23-H1 lies in pathways that it interrupts via binding and inactivation of proteins.

OBJECTIVE: To examine the genomic status and the mRNA expression of the nm23-H1 gene, and to analyse the relationship between metastasis of non-small cell lung carcinomas and the gene abnormalities. METHOD: By using PCR-SSCP and semiquantitative RT-PCR, the nm23-H1 gene mutation and mRNA expression were studied in 31 cases of non-small cell lung cancer. The normal tissue adjacent to carcinoma and normal lung tissue were used as control. RESULT: None of the nm23-H1 gene mutation was found in all the lung cancer samples. Decreased expressions of this gene were found in 14 of 20 lung cancer cases with lymph node metastasis, the rate (14/20) of this was significantly higher than that (3/11) of the non-metastatic cancers (P < 0.05). CONCLUSION: These data indicate that nm23-H1 may be a putative metastasis suppressor gene which had shown reverse regulating activation in the metastatic progression of lung cancers, and the level of nm23-H1 mRNA may be considered as one of the pathological indicators in predicting metastatic potential of lung cancers.

Lung cancer is the leading cause of cancer death in both men and women. tumor metastasis is an essential aspect of lung cancer progression. nm23-H1 is a metastasis suppressor gene. The molecular mechanism by which nm23-H1 suppresses the metastasis is still unclear. Here, we compared the gene expression profile of human large cell lung cancer cell line NL9980 by nm23-H1 gene silencing with that of negative control cells to comprehensively investigate nm23-H1-mediated changes in gene expression of NL9980 cells. Microarray assay revealed that expression of 733-known genes (1.9%, 733/38,500) were altered in response to nm23-H1 gene silencing, including 466 upregulated genes and 267 downregulated. real-time PCR assay of the expression changes indicated that 81.82% (45/55) of verified genes were consistent with that observed in microarray assay. The upregulated genes included MMP-1, -2, SNAI2, CXCL1, 2, 3, PAI-2, while the downregulated genes included cystatin B, TIMP-2, E-cadherin, centrin-2, all of which have been associated with tumor metastasis. Furthermore, we confirmed by Western blot that the expression of MMP-1 and -2 were significantly increased while that of cystatin B was dramatically decreased in NL9980-nm23-H1 silencing cells. The NL9980-nm23-H1 silencing cells exhibited significantly more S phase growth and invasive ability. Thus, silencing of nm23-H1 gene caused metastasis-related gene expression changes in lung cancer cells. The knockdown of nm23-H1 expression may change the lung cancer cells to a more invasive phenotype through alteration in the expression of a set of genes.

BACKGROUND: To analyze the prognostic significance of the genes casein kinase 2 alpha subunit (CSNK2A1), anti-apoptosis clone-11 (AAC-11), and tumor metastasis suppressor NME1 in completely resected non-small cell lung cancer (NSCLC) patients. MATERIAL/METHODS: Total RNA was extracted from 145 cases of completely resected, formalin-fixed, paraffin-embedded NSCLC tissues. mRNA expression levels of CSNK2A1, AAC-11, and NME1 were detected by real-time reverse-transcriptase polymerase chain reaction. Univariate and multivariate survival analyses were used to identify factors related to prognosis. RESULTS: A correlation between CSNK2A1 and AAC-11 mRNA expression levels (rs=0.366, p=0.000) was found. Univariate analysis showed that high expression of CSNK2A1 and AAC-11 was predictive of poor prognosis in NSCLC patients (p=0.029 and 0.044, respectively), especially when expression levels of both genes were concomitantly high (p=0.007). Multivariate Cox regression analysis showed that high expression of CSNK2A1, or concomitantly high expression of CSNK2A1 and AAC-11, are independent prognostic factors of poor survival in NSCLC patients. However, NME1 mRNA expression level did not significantly influence survival in NSCLC patients. CONCLUSIONS: This retrospective study indicates that CSNK2A1 and AAC-11, especially in combination, are useful prognosis markers in NSCLC patients after complete resection, independent of lymph node metastasis status.

BACKGROUND: It has been confirmed that nm23-H1 gene is one of the tumor metastasis suppressor genes. Up to now, the exact mechanism of nm23-H1 gebe is uncertain. The aim of this study the mechanism of metastasis suppressor gene nm23-H1 involving in the Ras signaling of lung cancer. METHODS: The wild and mutant type of pEGFP-nm23-H1 plasmids [WT (wild type), H118F, S120G, P96S, S44A] were transfected into the L9981 lung cancer cell lines through liposome method, and the complex of KSR and nm23-H1 was detected through co-immunoprecipitation and Western blot assay. RESULTS: The human KSR could be detected in the nm23-H1 immunoprecipitations in all the trasfected L9981 lung cancer cell lines. But no significant difference of KSR expression was found in the wild and mutant nm23-H1 trasfected cell lines (F =0.190, P =0.938). CONCLUSIONS: There was a close interaction between nm23-H1 and KSR, which was independent of the nm23-H1 mutation. Nm23-H1 involving in the Ras signaling of lung cancer may be through the KSR gene.

OBJECTIVE: To study on invasion and metastasis-associated genes of lung cancer related with NM23-H1 gene. METHODS: Human gene expression chip based on the subtracted cDNA libraries was constructed. After microarray hybridization, clones sequencing, sequence homology search, the information of differently expressed genes in human large cell lung cancer cell line of L9981 and L9981-nm23-H1 were obtained and then further confirmed by real-time quantitative PCR. RESULTS: Gene expression profiling chips of differently expressed genes in human large cell lung cancer cell line L9981 and L9981-nm23-H1 were successfully constructed. After microarray hybridization, sequence homology search, 19 differentially expressed genes were observed. After real-time quantitative PCR evaluation, we found that the mRNA of 8 genes including PSMA7, SBDS, ODC1, YARS, CSDA, PTP4A1, SHPRH and TOMM7 was up-regulated in the cell line of L9981 after transfected with NM23-H1 gene, whereas the mRNA of PKM2 and GMNN was down-regulated. CONCLUSION: NM23-H1 gene may be the upstream regulator of metastasis-associated genes, which can regulate the downstream genes to achieve a series of lung cancer metastatic potential.

Persistent STAT3 activation is a critical event in tumorigenesis and metastatic progression. Recent studies have found higher levels of STAT3 in metastatic tissues than in primary tumor tissues. We speculated that such increased STAT3 activity might be attributed to a loss of function or reduction in expression of metastasis inhibitory protein during cancer progression, and we therefore examined the role of tumor metastasis-suppressor nm23-H1 in the activation of STAT3 in the A549 lung cancer cell line. We found that IL-6-dependent induction of tyrosine phosphorylation and activation of STAT3 were influenced by nm23-H1 inhibition. IL-6-induced STAT3(Tyr705) phosphorylation was significantly enhanced in A549 cells transfected with siRNA specific for nm23-H1, and the effect of nm23-H1 depletion on IL-6-induced STAT3(Tyr705) phosphorylation was reversed by ectopic expression of shRNA-resistant nm23-H1 protein. Moreover, STAT3 directly bound to the STAT3 binding site on the nm23-H1 promoter and activated its expression. Thus, we have identified a new feedback mechanism that might provide insight into an in-built metastasis-suppression function in tumor cells and which could be a logical new target for treatment of early metastatic disease.

The nm23 gene, a putative metastasis suppressor gene, was originally identified by its reduced expression in highly metastatic K-1735 murine melanoma cell lines, as compared to related, low metastatic melanoma cell lines. Transfection of nm23 cDNA has been reported to suppress malignant progression in Drosophila and mammalian cells. Highly conserved homologues of nm23 have been found in organisms ranging from the prokaryote Myxococcus xanthus to Drosophila, where the gene is involved in normal development and differentiation. The product of the nm23 gene exhibits a nucleoside diphosphate kinase activity, yet the nucleoside diphosphate kinase activity of Nm23 does not correlate with its apparent biological functions. We review recent cellular, genetic, biochemical and X-ray crystallographic data to formulate and evaluate hypotheses concerning the molecular mechanism of nm23 action.

The putative metastasis suppressor genes, NME1(nm23-1) and NME2(nm23-2), were examined in a model system we developed to approximate the dissemination of melanoma from a primary skin tumor. We utilized two autologous human melanoma cell lines, IV Cl 1 and IV Cl 3, which displayed qualitatively different metastatic phenotypes following subdermal inoculation into nude mice. Highly metastatic IV Cl 1 cells expressed approximately 5 fold lower levels of protein encoded by NME genes than non-metastatic IV Cl 3 cells. Similar differences in NME protein levels were observed in tumors induced by the two cell lines in nude mice. There were no differences in NME mRNA levels between these two cell lines, suggesting that expression of these proteins is regulated at a post-transcriptional level. We found a ser122-pro mutation in the NME2 gene of metastatic IV Cl 1 cells. A similar ser120-gly mutation in NME1 has been found in human neuroblastoma, suggesting that mutation in this region may be a general phenomenon related to tumor progression. These mutations may have functional consequences since they eliminate potential phosphorylation sites and may affect the tertiary structure of mature protein complexes.

The nm23 gene is a potential metastasis-suppressor gene originally identified in a murine melanoma line. Several investigators have reported the probable inverse association of nm23 expression with disease prognosis and/or metastasis. Since there are now 2 known isotypes of human nm23, namely nm23-HI and -H2, we immunohistochemically examined expression of these isotypes in human breast-cancer tissues using monoclonal antibodies (MAbs) specific for each isotype protein. We also analyzed expression of c-erbB-2 in the same collection of cancer tissues, in order to examine the significance of nm23 expression in comparison with c-erbB-2 expression. Of 130 tumors from breast-cancer patients, 73 (56%) and 69 (53%) positively expressed nm23-HI and -H2 respectively. expression of c-erbB-2 was positive in 36 (28%). expression of nm23-HI, but not nm23-H2, was inversely associated with lymph-node metastasis (p < 0.01). expression of c-erbB-2 was associated with Tnm stage, tumor size and lymph-node metastasis (p < 0.01, p < 0.05 and p < 0.05 respectively). Overall survival was better (p = 0.014) in patients in whom expression of nm23-HI was positive than in those in whom it was negative. In multivariate analyses using a Cox's proportional-hazards regression model with 9 variables, nm23-HI showed the fourth greatest contribution to patient survival following lymph-node metastasis, Tnm stage and menopausal status. No significant contribution was shown for c-erbB-2 expression. nm23-HI, but not nm23-H2, may perform a role in disease prognosis in addition to its participation in cancer metastasis. It may have value for predicting long-term survival of human breast-cancer patients.

NM23 is a putative metastasis-suppressor gene for some human cancers. Here we have studied NM23 expression during melanoma progression using Northern blotting and immunocytochemistry. There was no significant difference in the average amounts of NM23 mRNA between cell lines derived from metastatic and primary melanomas. The level of NM23 mRNA was also determined for three pairs of poorly metastatic parental (P) and their highly metastatic variant (M) cell lines; the ratios for M/P were 1.2, 0.98 and 0.80. Next we used immunocytochemistry to study NM23 protein in normal skin, benign naevi and primary and metastatic melanomas. Melanocytes in all normal skin and benign samples were positive for NM23; however most primary melanomas (7/11) were not stained by the antibody. All metastatic melanoma samples (5/5) were positively stained. Findings were similar with an antiserum reactive with both forms of NM23 (H1 and H2), and with an antibody specific for NM23-H1. No relationship was apparent between NM23 immunoreactivity in primary tumours and their aggressiveness or prognosis. Hence, in contrast to the situation described for murine melanoma, the amount of NM23 mRNA or protein in human melanoma did not correlate inversely with metastasis.

An inverse correlation between the nm23 RNA level and tumour progression of melanocytes has been reported. To elucidate whether the expression of nm23 gene product in malignant melanoma is also inversely correlated with metastatic potential, conventional prognostic parameters or the tumour suppressor protein p53, immunohisto-chemical studies using a monoclonal antibody against nm23-H1 protein were performed on 138 benign and malignant melanocytic tumours. The expression of nm23 protein was compared with that of p53 protein and conventional clinicopathological prognostic factors. The nm23 protein level in benign melanocytes and metastatic melanoma cells was also studied by Western blot analysis. No significant difference regarding the protein was observed between naevi and melanomas, either at histological or protein levels. The expression correlated with local recurrence within 1 year after surgery, level of invasion and tumour thickness, but no parallels were observed between the nm23 and p53 proteins, suggesting that gene is regulated by independent mechanisms, although located on the same chromosome. There was no inverse correlation between the nm23 protein and melanoma metastasis which suggested that the nm23 protein does not appear to be lost during melanoma metastasis.

The nm23 gene is a potential metastasis suppressor gene originally identified using a murine melanoma cell line. The nm23 proteins are nucleoside diphosphate (NDP) kinases, and there are two isotypes in human and other mammalian cells. Immunohistochemical analysis with anti-rat NDP kinase (NDP kinase alpha, NDP kinase beta) monoclonal antibodies was performed in deparaffinized sections of 56 specimens of oral squamous cell carcinoma. Detection of these NDP kinases in tissue samples was correlated with clinical and histopathological factors. The NDP kinase A (nm23 H1 protein)-positive group was apt to have a lower frequency of neck metastasis than the negative one (p < 0.01). Consequently, it is suggested that NDP kinase A might be a metastasis suppressor factor that may be useful for predicting tumor metastases in patients with oral squamous cell carcinoma.

The nm23 gene was originally identified in murine melanoma cell lines as a putative metastasis suppressor gene. 1a a limited number of studies in breast carcinomas nm23 mRNA and/or protein levels were found to correlate inversely with lymph node metastases, and positively with the survival of patients. Using a monoclonal antibody to nm23-Hl protein we have examined the immunohistochemical expression of nm-23 in breast ductal carcinomas of 44 lymph node-negative patients with similar tumor pathologic features. The mean follow-up period of the patients was 138 months. Thirty two out of 44 tumors (72%) disclosed high immunohistochemical expression of nm23 protein and 12 (28%) low or negative expression. No correlation was observed between nm23 expression and the relapse or death rate of the patients. Similarly, no association was found between nm23 protein levels and estrogen receptor status or p53 protein. Our results do not seem to agree with the proposed antimetastatic property of nm23 protein, and indicate that its immunohistochemical determination has no prognose significance in the management of node-negative breast cancer patients.

Uveal melanoma is characterized by an unpredictable clinical course, during which metastatic disease may occur after a prolonged and, at present, undefinable disease-free interval. Because of its relative rarity and the dispersion of cases, the possible genetic alterations implicated in the invasive and metastatic behaviour of this ocular neoplasm have not yet been characterized. The aim of this immunohistochemical retrospective study was to assess the expression of nm23 gene product, proposed to be a metastasis-suppressor gene, in uveal melanoma and to analyse its prognostic significance in relation to the various conventional histopathological parameters, currently considered the major prognostic indicators in this intra-ocular neoplasm. We analysed formalin-fixed paraffin-embedded samples excised from 33 patients with uveal melanoma. Of these, 22 (67%) were positive for monoclonal antibody nm23. This nm23 positivity was inversely associated with scleral invasion level (P = 0.001) and largest tumour diameter (P = 0.02), which represent the two most significant prognostic factors for metastasis. On the other hand, there was no correlation between nm23 expression and other prognostic markers such as cell type, intraocular location or clinical characteristics. These results may suggest a close relationship between nm23 gene expression and metastatic potential of uveal melanomas. In addition, analysis of nm23 gene expression on bioptic tissue may represent an extreme useful prognostic tool for metastatic progression of uveal melanomas.

The nm23 gene is a potential metastasis suppressor gene originally identified using a murine melanoma cell line. Immunohistochemical investigation on the role of nm23 gene product (nucleoside diphosphate kinase: NDPK) in squamous cell carcinomas of the oral cavity was performed and compared to clinical and histopathological factors. The stainability of NDPK did not correlate to tumour extent, differentiation and mode of invasion. However, the NDPK positive group tended to have a lower frequency of lymph node metastasis, and a better prognosis than the NDPK negative group. Consequently, nm23/NDPK is suggested to be a metastasis suppressor factor that may be useful for predicting tumour metastasis and prognosis in oral squamous cell carcinoma.

The nm23 gene is a potential metastasis suppressor gene originally identified using a murine melanoma cell line. The expression of nm23-H1 protein was examined immunohistochemically in 50 eligible patients with esophageal squamous cell carcinoma (ESCC). The expression was not correlated with other prognostic factors including lymph node metastases; however, overall survival rates of nm23-H1-negative patients were significantly shorter than those of nm23-H1-positive patients (P < 0.05). Furthermore, reduced expression of nm23-H1 was associated with shorter overall survival in patients with involved lymph nodes (P < 0.01), but not in patients without involved lymph nodes. These data support the conclusion that reduced expression of nm23-H1 may be associated with poor prognosis of ESCC patients, suggesting the value of nm23-H1 expression as a prognostic marker for ESCC patients, especially ESCC patients with involved lymph nodes.

NM23 is considered to be a metastasis suppressor gene the role of which as prognosticator in the case of malignant skin melanoma (MM) is highly controversial due to different results on gene, and protein expressions. Accordingly, we analyzed the protein expression of NM23 in 32 primary skin melanomas with a follow-up period of 5 years minimum. We found that NM23 expression was the lowest in the thickest primary tumors (based on the % of the positive cells and the incidence of low expressor tumors) but the difference was not significant statistically due to the extreme heterogeneity of the tumors. When primary tumors were grouped according to their biological behavior (non-metastatic; lymph-node (LN) metastatic; organ and LN metastatic tumors) we observed that the lowest NM23 protein expression (based on the % of positive tumor cells as well as on the incidence of low expressor tumors) was found in the LN metastatic tumors compared to other groups (p < 0.05). The NM23 phenotype of the primary tumors remained stable in the corresponding LN metastases in the case of the 5 analyzed tumors. There was no difference in the 5-year survival between patients with low (< 50% positive cells) or high NM23 protein expressing primary tumors. Collectively, these data suggest that the NM23 protein expression in the primary tumors of MM predicted lymphatic spread but did not affect 5-year survival because it did not correlate with organ metastasis.

The human nm23-H1 was discovered as a tumor metastasis suppressor based on its reduced expression in melanoma cell lines with low versus high metastatic potential. It encodes for one of two subunits of the nucleoside-diphosphate kinase. Besides its role in the maintenance of the cells NTP pool, nm23 plays a key role in different cellular processes. The role of nm23-H1 in these processes still has to be elucidated. Our goal was to identify Nm23-H1 downstream targets by subjecting Nm23-H1 overexpressing CAL 27 cells oral squamous cell carcinoma (OSSC) to microarray analysis. The genes with changed expression patterns could be clustered into several groups: transforming growth factor beta (TGFbeta) signaling pathway, cell adhesion, invasion and motility, proteasome machinery, cell-cycle, epithelial structural and related molecules and others. Based on the expression patterns observed we presume that nm23-H1 might have a role in OSSCs, which should be confirmed by future experiments.

BACKGROUND: Some dermatopathological parameters are recognized as dominant indicators of high metastatic potential in melanoma, especially Breslow thickness, ulceration, Clark's level of invasion and mitotic rate. Because NM23 protein is the product of a melanoma metastasis suppressor gene, the aim of this study was to compare such dermatopathological indicators of melanoma prognosis with NM23 protein expression in primary cutaneous melanoma. METHODS: The immunohistochemical NM23 expression was semiquantitatively assessed in 30 primary cutaneous melanomas. Ten dermatopathological parameters were evaluated and compared with NM23 expression. RESULTS: A significant inverse correlation was found for NM23 expression in comparison with Breslow thickness (p < 0.01), ulceration (p < 0.05), Clark's level (p < 0.01), mitotic rate (p < 0.05), and vertical growth phase (p < 0.05). By contrast, no significant correlation was found for NM23 expression in comparison with cell morphology, presence of adjacent nevus, pigmentation, tumor-infiltrating lymphocytes, and regression was impossible to evaluate. CONCLUSIONS: The expression of NM23 protein in primary cutaneous melanoma is significantly inversely correlated with dermatopathological parameters currently recognized as powerful indicators of melanoma prognosis. NM23 may be therefore considered in the dermatopathological evaluation of primary cutaneous melanoma.

In humans, NM23-H1 is a metastasis suppressor whose expression is reduced in metastatic melanoma and breast carcinoma cells, and which possesses the ability to inhibit metastatic growth without significant impact on the transformed phenotype. NM23-H1 exhibits three enzymatic activities in vitro, each with potential to maintain genomic stability, a 3'-5' exonuclease and two kinases, nucleoside diphosphate kinase (NDPK), and protein histidine kinase. Herein we have investigated the potential contributions of NM23 proteins to DNA repair in the yeast, Saccharomyces cerevisiae, which contains a single NM23 homolog, YNK1. Ablation of YNK1 delayed repair of UV- and etoposide-induced nuclear DNA damage by 3-6h. However, YNK1 had no impact upon the kinetics of MMS-induced DNA repair. Furthermore, YNK1 was not required for repair of mitochondrial DNA damage. To determine whether the nuclear DNA repair deficit manifested as an increase in mutation frequency, the CAN1 forward assay was employed. An YNK1 deletion was associated with increased mutation rates following treatment with either UV (2.6x) or MMS (1.6 x). mutation spectral analysis further revealed significantly increased rates of base substitution and frameshift mutations following UV treatment in the ynk1Delta strain. This study indicates a novel role for YNK1 in DNA repair in yeast, and suggests an anti-mutator function that may contribute to the metastasis suppressor function of NM23-H1 in humans.

nm23-h1 was the first metastasis suppressor gene to be identified in humans, with early studies demonstrating its ability to inhibit the metastatic potential of breast carcinoma and melanoma cell lines. This report outlines recent findings from our laboratory indicating that the metastasis suppressor function of NM23-H1 in human melanoma involves a spectrum of molecular mechanisms. Analysis of NM23-H1-dependent profiles of gene expression in human melanoma cell lines has identified a host of target genes that appear to mediate suppression of directional motility. Of particular interest is a subset of motility-suppressing genes whose regulation by NM23-H1 is independent of its known kinase and 3'-5' exonuclease activities. In parallel, we have recently observed that NM23-H1 expression appears to be required for genomic stability and for optimal repair of DNA damage produced by ultraviolet radiation and other agents. Thus, NM23-H1 might oppose not only the motile and invasive characteristics of metastatic cells but also the acquisition of mutations that drive malignant progression to the metastatic phenotype itself.

Nm23-H1, also known as NDPK-A, was the first of a class of metastasis suppressor genes to be identified. Overexpression of Nm23-H1 in metastatic cell lines (melanoma, breast carcinoma, prostate, colon, hepatocellular, and oral squamous cell carcinoma) reduced cell motility in in vitro assays and metastatic potential in xenograft models, without a significant effect on primary tumor size. The mechanism of Nm23-H1 suppression of metastasis, however, is incompletely understood. Nm23-H1 has been reported to bind proteins, including those in small G-protein complexes, transcriptional complexes, the Map kinase, the TGF-beta signaling pathways and the cytoskeleton. Evidence supporting these associations is presented together with evidence of resultant biochemical and phenotypic consequences of association. Cumulatively, the data suggest that part of the anti-metastatic function of Nm23-H1 lies in pathways that it interrupts via binding and inactivation of proteins.

The Nm23 gene is a metastatic suppressor identified in a melanoma cell line and expressed in different tumors where their levels of expression are associated with reduced or increased metastatic potential. Nm23 is one of the over 20 metastasis suppressor genes (MSGs) confirmed in vivo. It is highly conserved from yeast to human, implying a critical developmental function. tumors with alteration of the p53 gene and reduced expression of the Nm23 gene are more prone to metastasis. Nm23-H1 has 3'-5' exonuclease activity. This review focuses on the role of Nm23 in cancer progression and also a potential novel target for cancer therapy.

Melanocytic nevus rests in lymph nodes are a known diagnostic challenge, especially in patients with a history of melanoma. Reticulin and NM23 have been studied in this context. The pattern of reticulin staining in melanomas surrounds groups/nests of melanocytes but individual cells in benign nevi. NM23, a metastasis-suppressor gene, has an association with metastatic potential in melanomas and some carcinomas. Twenty-eight cases (14 cases of metastatic melanoma to lymph nodes and 14 cases of lymph node nevus rests, all confirmed with Melan-A staining) were stained with reticulin and NM23. The pattern of reticulin staining was reported as surrounding groups if staining was noted in approximately 5-10 melanocytes in greater than 50% of the lesion but was otherwise reported as surrounding individual melanocytes. Cytoplasmic staining was considered to represent reactivity for NM23. Reticulin staining around groups of melanocytes was identified in all 14 cases of metastatic melanoma. Regarding nodal nevus rest cases, 12 of 14 cases (86%) demonstrated staining around individual melanocytes, whereas in 2 cases, reticulin surrounded melanocytic groups. NM23 staining was equivocal in all cases. Reticulin staining reliably invests groups of melanocytes in cases of metastatic melanoma, whereas in nodal nevus rests, it predominantly surrounds individual melanocytes. NM23 demonstrated no discriminatory value in this analysis. In cases in which a collection of melanocytes is present within a lymph node, reticulin deposition around individual melanocytes supports a diagnosis of lymph nodal nevus rest.

tumor metastasis is responsible for a high degree of mortality in cancer patients. One of the genes involved in tumor metastasis is NM23. At present, eight human isoforms, transcribed from different NM23 genes, have been detected. The gene products have been identified as nucleoside diphosphate kinases (NDPKs), most of which catalyse the transfer of the gamma-phosphate of a (deoxy)nucleoside triphosphate to a (deoxy)nucleoside diphosphate. However, the function of NDPK isoforms involved in tumor metastasis cannot be explained on the basis of their phosphotransferase activity alone. At present, several other properties, like transcriptional regulation and protein kinase activity, have been assigned to these proteins. Moreover, it has also been shown that NDPKs interact with several other proteins, and binding partners of NDPKs are identified at an increasing rate. Accumulating evidence indicates that protein-protein interactions modulate the molecular action of NDPKs. In this review we provide a brief overview of how NDPKs are correlated with cancer, and discuss when and how the activities assigned to NDPKs may affect metastasis, with special emphasis on the role of protein-NDPK interactions in this process.

The protein product of nm23-H1 gene has activity of nucleoside diphosphate (NDP) kinase, which catalyzes the phosphorylation of nucleoside diphosphates to the corresponding nucleoside triphosphates. Reductions in nm23 expression have been significantly associated with aggressive behavior in melanoma, breast, colon, and gastric carcinomas. On the contrary, high levels of nm23 gene expression are noted in the advanced stage of thyroid carcinomas and associated with significant reductions in survival for neuroblastoma and osteosarcoma patients. Although expression of nm23/NDP kinase is divergent in various malignant tumors, its reduced expression seems to be related to increased metastatic potential in most carcinoma types. However, it is hypothesized that nm23 may play a tissue-specific role, and that different regulatory mechanisms may act in different tumors. In ovarian carcinoma, nm23-H1/NDP kinase may be correlated with some clinicopathologic characteristics. In cervical cancer, nm23-H1 is probably involved in cervical carcinogenesis and correlated with some aggressive parameters. Overexpression of nm23-H1 protein may indicate poor survival for cervical cancer patients. Other than histidine 118 residue (amino acid sequence 118: histidine) concerned with NDP kinase activity of nm23-H1, serine 120 (amino acid sequence 120: serine) related activity of histidine-dependent protein phosphotransfer was recently reported to be responsible for its biological suppressive effects. To inhibit metastatic potential, nm23-H1 is also demonstrated to co-immunoprecipitate the kinase suppressor of Ras and phosphorylate it, and therefore reduce activation of the extracellular signal-regulated kinase mitogen-activated protein kinase pathway in response to signaling.

Cytoskeleton rearrangement is necessary for tumor invasion and metastasis. Cellular molecules whose role is to regulate components of the cytoskeletal structure can dictate changes in cellular morphology. One of these molecules is the suppressor of tumor metastasis Nm23-H1. The level of Nm23-H1 expression has been linked to the invasiveness and metastatic potential of human cancers including melanoma and breast cancer. In this report, we demonstrate an interaction between the suppressor of tumor metastasis Nm23-H1, and Dbl-1, an oncoprotein which is associated with guanine exchange and belongs to a family of Guanine Exchange Factors (GEF). Nm23-H1 also was shown to bind pDbl which is the proto-oncoprotein of Dbl. Interestingly, the interaction between Nm23-H1 and Dbl-1 rescues the suppression of the cell motility activity Nm23-H1. Moreover, this interaction results in loss of the ability of the Dbl-1 oncoprotein to function as a GEF for the critical Rho-GTPase family member Cdc42. The loss of GTP loading onto Cdc42 resulted in a dramatic reduction in adhesion stimulated ruffles and suggests that Nm23-H1 can negatively regulate cell migration and tumor metastasis by modulating the activity of Cdc42 through direct interaction with Dbl-1.

The DHH superfamily human protein h-prune, a binding partner of the metastasis suppressor nm23-H1, is frequently overexpressed in metastatic cancers. From an evolutionary perspective, h-prune is very close to eukaryotic exopolyphosphatases. Here, we show for the first time that h-prune efficiently hydrolyzes short-chain polyphosphates (k cat of 3-40 s (-1)), including inorganic tripoly- and tetrapolyphosphates and nucleoside 5'-tetraphosphates. Long-chain inorganic polyphosphates (>or=25 phosphate residues) are converted more slowly, whereas pyrophosphate and nucleoside triphosphates are not hydrolyzed. The reaction requires a divalent metal cofactor, such as Mg (2+), Co (2+), or Mn (2+), which activates both the enzyme and substrate. Notably, the exopolyphosphatase activity of h-prune is suppressed by nm23-H1, long-chain polyphosphates and pyrophosphate, which may be potential physiological regulators. Nucleoside triphosphates, diadenosine hexaphosphate, cAMP, and dipyridamole (inhibitor of phosphodiesterase) do not affect this activity. mutation of seven single residues corresponding to those found in the active site of yeast exopolyphosphatase led to a severe decrease in h-prune activity, whereas one variant enzyme exhibited enhanced activity. Our results collectively suggest that prune is the missing exopolyphosphatase in animals and support the hypothesis that the metastatic effects of h-prune are modulated by inorganic polyphosphates, which are increasingly recognized as critical regulators in cells.

tumor invasion and metastasis is regulated by a number of cellular molecules known to be involved in signaling and cytoskeletal rearrangement. One of these molecules is the suppressor of tumor metastasis Nm23-H1 which linked to invasiveness and metastatic potential of human cancers. Nm23-H1 expression is down-regulated in human melanoma and invasive breast carcinoma. Recent studies have shown an association between the Nm23-H1 and oncoprotein Dbl-1 which is associated with guanine exchange and belongs to a family of Guanine Exchange Factors (GEF). In this report we show a direct interaction in vitro and in human B cells and specifically identified the pleckstrin homology domain of Dbl-1 as the domain which binds to Nm23-H1. Furthermore, Nm23-H1 and Dbl-1 colocalized in the cytoplasm of COS-7 cells when expressed exogenously and showed predominant signals at the periphery of the cells particularly at the plasma membrane. Interestingly, Dbl-1 and Cdc42 expression rescued the suppressive activities of Nm23-H1 in cell migration assays. We show that Cdc42 a regulatory protein involved in cytoskeletal reorganization, cell growth and development can bind to Nm23-H1 and the kinase deficient mutant H118F but only weakly to the mutant P96S which lacks the ability to suppress cell migration and metastasis. Cdc42 also colocalized with Nm23-H1 and the Dbl-1 proteins as specific punctate signals in the cytoplasm and at the cell membrane. Nm23-H1 also lead to the reduction in membrane ruffles and protuberances when expressed with Dbl-1 and Cdc42. Surprisingly, Nm23-H1 interacted with Cdc42 as well as Rac1 but somewhat weaker with RhoA. These studies suggests that Nm23-H1 can negatively regulate cell migration and tumor metastasis by modulating the activity of Cdc42 and possibly other Rho family members through interaction with Dbl-1.

Chromosomal instability and the subsequent genetic mutations are considered to be critical factors in the development of the majority of solid tumors. Here, we describe how the nucleoside diphosphate kinase Nm23-H1, a protein with a known link to cancer progression, regulates a critical step during cytokinesis. Nm23-H1 acts to provide a local source of GTP for the GTPase dynamin. Loss of Nm23-H1 in diploid cells leads to cytokinetic furrow regression, followed by cytokinesis failure and generation of tetraploid cells. Loss of dynamin phenocopies loss of Nm23-H1, and ectopic overexpression of WT dynamin complements the loss of Nm23-H1. In the absence of p53 signaling, the tetraploid cells resulting from loss of Nm23-H1 continue cycling and develop classic hallmarks of tumor cells. We thus provide evidence that the loss of Nm23-H1, an event suspected to promote metastasis, may additionally function at an earlier stage of tumor development to drive the acquisition of chromosomal instability.

IKKbeta functions as a principal upstream activator of the canonical NF-kappaB pathway by phosphorylating IkappaB, leading to its proteasomal degradation. Because IKKbeta is considered a therapeutic target, understanding its regulation may facilitate the design of efficient regulators of this molecule. Here, we report a novel IKKbeta-interacting molecule, NME1L, a splicing variant of the NME1 protein. NME1 has attracted attention in cancer research because of its antimetastatic activity and reduced expression in multiple aggressive types of cancer. However, the effect was just moderate but not dramatic in anti-cancer activities. We found that only NME1L interacts with IKKbeta. Exogenous expression of NME1L resulted in a potent decrease in TNFalpha-stimulated NF-kappaB activation, whereas knockdown of NME1/NME1L with shRNA enhanced activity of NF-kappaB. NME1L down-regulates IKKbeta signaling by blocking IKKbeta-mediated IkappaB degradation. When NME1L was introduced into highly metastatic HT1080 cells, the mobility was efficiently inhibited. Furthermore, in a metastasis assay, NME1L-expressing cells did not colonize the lung. Based on these results, NME1L is a potent antimetastatic protein and may be a useful weapon in the fight against cancers.

Using the specific monoclonal antibody of nm23H1 gene product and the immunohistochemical technique, we studied the expression of DNPK/nm23H1 and its correlation with lymph-node metastasis in nasopharyngeal carchnoma (NPC). The results revealed that, in a total of 31 cases of NPC, 13 showed positive expression with a positive rate of 41.9%. Among 21 cases of NPC with no lymph-node metastasis, 11 (52.3%) showed positive expression while only 20% (2/10) of those with lymph-node metastasis were positive, and there was significant difference between them (P < 0.01). Meantime the expression was very low or not detectable in the cancerous cells of the match lymph-node metastasis and all 10 cases showed negative expression. Statistical significant difference was found between the group of NPC with no metastasis and the group of lymph-node metastasis (P < 0.01). The above results indicated that high expression of DNPK/nm23H1 existed in the group of NPC with no metastasis while low expression existed in those with metastasis and lymph-node metastasis. It suggests that the expression of nm23H1 is inversely associated with lymph-node metastasis. The DNPK/nm23H1 may play a role in polymerization of microtubulin protein and participate in the process of cancer metastasis.

OBJECTIVE: To investigate the relationship between the expressions of KAI1, nm23, ETS-1, vascular endothelial growth factor (VEGF) and microvascular density (MVD) and lymph node metastasis and prognosis in nasopharyngeal carcinoma (NPC). METHODS: The Envision immunohistochemical method was used to detect the expressions of KAI1, nm23, ETS-1 and VEGF in 50 cases of non-keratinizing carcinoma (NKC) with cervical lymph node metastasis, 30 cases of NKC without cervical lymph node metastasis at the primary diagnoses and 30 cases of non-tumor nasopharyngeal tissues (NP). The microvascular density was counted by immunostaining with CD34. RESULTS: (1) The expression rates of KAI1 and nm23 protein in NKC with cervical lymph node metastasis group and without cervical lymph node metastasis group and NP group increased successively , the difference being significant (P < 0.05); The expression rates of ETS-1 and VEGF protein in NKC with cervical lymph node metastasis group and without cervical lymph node metastasis group and NP group increased successively, the difference being significant (P < 0.05). (2) In 80 NKC cases, the MVD was respectively lower in KAI1 and nm23 protein positive groups than those in the negative groups (P < 0.05); the MVD was respectively higher in ETS-1 and VEGF protein positive groups than those in the negative groups (P < 0.05 ). (3) There was significant difference between the MVD, the number of NKC without cervical lymph node metastasis cases in the single expression of KAI1 or nm23 protein and in common expression of KAI1 and nm23 protein (P < 0.05), in the same as between the single expression of ETS-1 or VEGF protein and in common expressions of ETS-1 and VEGF protein (P < 0.05). (4) There was positive correlation between the expressions of KAI1 and nm23 protein (P < 0.01), as well as between the expressions of ETS-1 and VEGF protein (P < 0.01). (5) the 5-year survival rates of the patients correlated with cervical lymph node metastasis and the expressions of KAI1, nm23, ETS-1 and VEGF proteins in NKC (P < 0.05). CONCLUSIONS: The expressions of KAI1, nm23, ETS-1 and VEGF proteins were highly related to MVD in NPC,cervical lymph node metastasis and prognosis. They might be considered to be reference indicator for evaluating the cervical lymph node metastasis and prognosis of NPC.

OBJECTIVE: To identify proteins associated with nasopharyngeal carcinoma (NPC) metastasis, and provide scientific basis for the prevention and cure of NPC. METHODS: A two-dimensional gel electrophoresis and mass spectrometry were performed to screen for differential proteins between highly metastatic 5-8F and non-metastatic 6-10B NPC cell lines. Western blot was used to confirm the differential proteins. We siRNA used to inhibit the expression of differential protein nm23-H1 to determine the association of nm23-H1 with NPC in vitro invasive ability. Immunohistochemistry and statistics were used to evaluate the correlation of nm23-H1 expression with clinicopathological features and clinical outcomes in paraffin-embedded archival tissues including 93 cases of primary NPC and 20 cases of cervical lymphonode metastatic NPC (LMNPC). RESULTS: A total of 15 differential proteins in the 2 cell lines were identified by a proteomic approach, and 3 differential proteins were selectively confirmed. Downregulation of nm23-H1 by siRNA significantly increased the in vitro invasive ability of 6-10B. Significant nm23-H1 downregulation was observed in LMNPC compared with primary NPC. nm23-H1 downregulation in primary NPC was positively correlated with lymphonode and distant metastasis, advanced clinical stage and recurrence. Survival curves showed that patients with nm23-H1 downregulation in primary NPC had a poor prognosis. Multivariate analysis confirmed that nm23-H1 expression level in primary NPC was an independent prognostic indicator. CONCLUSION: nm23-H1 behaves as a metastasis suppressor in NPC, and nm23-H1 downregulation in the is a biomarker for poor NPC prognosis.

BACKGROUND: Regional metastasis is an important factor in the treatment and prognosis of patients with head and neck squamous cell carcinoma. Although in recent years imaging techniques have improved, it is still impossible to detect small metastatic deposits. metastasis is mainly determined by properties of the primary tumor and its interaction with surrounding structures. OBJECTIVE: To identify markers that predict the presence of metastasis based on the features of the primary tumor. DESIGN: Correlation of the results of histological, immunohistochemical, and molecular biological analysis with clinical and histopathological data. MATERIALS AND METHODS: Several histological features and biological markers were examined in 31 laryngeal carcinomas. The following markers were selected on their putative role in the process of metastasis and were studied using immunohistochemical and/or Southern blot techniques: proliferating cell nuclear antigen (PCNA), p53, retinoblastoma tumor-suppressor gene (Rb), myc, bcl-2 (inhibitor of apoptosis), epidermal growth factor (EGF), EGF-receptor (EGFR), neu, nm23 (also known as NME1, putative metastasis suppressor), desmoplakin, neuron cell-adhesion molecule (N-CAM), epithelial cell-adhesion molecule (Ep-CAM), E-cadherin, cyclin D1 (CCND1), and EMS1. RESULTS: The presence of an inflammatory reaction surrounding the tumor (P = .07), eosinophilic infiltration (P = .16), positive immunostaining for Rb (P = .02), negative immunostaining for Ep-CAM (P = .13), and amplification of CCND1 and EMS1 (P = .05) correlated with nodal metastasis. The combination of an inflammatory reaction, eosinophilic infiltration, and staining for Rb and Ep-CAM resulted in a superior accuracy in assessing nodal metastasis. CONCLUSIONS: These results indicate that it is possible to predict and exclude lymph node metastasis by studying the features of the primary tumor only. When these results are confirmed in a larger series, biological markers may be powerful diagnostic tools with great impact on clinical decision making.

tumor suppressor genes that reduce metastatic potential have been described in a variety of different tumor types. One of the main tumor metastasis suppressor genes is nm-23, which is a nucleoside diphosphate kinase. Two isotypes, nm-23H1 and nm-23H2, have been cloned and map to chromosome 17q21.3. In a variety of tumors, including colon cancer and breast cancer, loss of expression of nm-23 is associated with lymph node metastasis. In other organ systems, however, this relationship is not seen. In head and neck squamous cell carcinomas (HNSCC), there have been conflicting results regarding the association between nm-23 protein expression and metastatic potential. To further explore the tumor metastasis suppressor function of nm-23 in HNSCC, we studied high-stage laryngeal carcinomas, tumors with and without cervical lymph node metastasis for nm-23 protein expression and loss of heterozygosity of the gene locus. Twenty-five cases were included (11 cases with and 14 cases without metastasis). Loss of heterozygosity for the nm-23 gene locus was seen in 7 of 22 (32%) informative tumors. Using immunohistochemistry, most tumors expressed nm-23, though decreased expression was seen in 10 of 25 (40%) cases. Only 2 tumors showed negative expression. We did not find a correlation between either protein expression or loss of heterozygosity with metastatic disease or any other adverse prognostic factors in this group of high-stage laryngeal squamous cell carcinomas. These data imply that nm-23 may be tumor suppressor gene involved in HNSCC but that it may not function as a tumor metastasis suppressor in high-stage laryngeal carcinoma.

The putative metastasis suppressor genes, NME1(nm23-1) and NME2(nm23-2), were examined in a model system we developed to approximate the dissemination of melanoma from a primary skin tumor. We utilized two autologous human melanoma cell lines, IV Cl 1 and IV Cl 3, which displayed qualitatively different metastatic phenotypes following subdermal inoculation into nude mice. Highly metastatic IV Cl 1 cells expressed approximately 5 fold lower levels of protein encoded by NME genes than non-metastatic IV Cl 3 cells. Similar differences in NME protein levels were observed in tumors induced by the two cell lines in nude mice. There were no differences in NME mRNA levels between these two cell lines, suggesting that expression of these proteins is regulated at a post-transcriptional level. We found a ser122-pro mutation in the NME2 gene of metastatic IV Cl 1 cells. A similar ser120-gly mutation in NME1 has been found in human neuroblastoma, suggesting that mutation in this region may be a general phenomenon related to tumor progression. These mutations may have functional consequences since they eliminate potential phosphorylation sites and may affect the tertiary structure of mature protein complexes.

Allelic deletions in the nm23, a metastasis suppressor gene, are known to occur in neuroblastomas, breast and colorectal carcinomas. Down-regulation of nm23 expression has been reported in various rodent and human tumor cells with high metastasis phenotype. Colorectal tumors showed overexpression of nm23. To elucidate the regulatory mechanisms of nm23, we isolated, cloned and sequenced the presumptive regulatory DNA fragment spanning the 5' region of the human nm23-H1 gene. The region's nucleotide sequence shows the presence of motifs typical for transcriptional elements such as TFIID, AP-1 and CTF/NF1. A common transcription initiation site is located at -136 upstream from the first ATG codon in placenta tissue, in breast, colorectal, prostate tumor cell lines and in primary colorectal tumor. Multiple transcription start sites were identified in tumor cell lines and colorectal tumor. When the promoter element was linked to a reporter gene, chloramphenicol acetyltransferase (CAT) and transfected in human 2fTGH cells, strong CAT activity was detected, which also showed that the presence of AP-1 and CTF/NF1 elements are essential for promoter activity. A detailed study of the structure and function of the promoter element of the nm23-H1 gene will help in understanding the regulatory mechanisms of nm23 expression and its role in tumor progression, especially in metastasis.

Nm23-H1 is a metastasis suppressor gene whose overexpression is associated with both reduced cell motility in various cancers and increased metastatic potential in neuroblastomas, osteosarcomas, and hematological malignances. We previously reported that Nm23-H1 exerts tumor suppressor action in prostate cancer cells and that h-Prune, which is overexpressed in various tumor types, binds Nm23-H1. Moreover, blockage of the Nm23-H1/h-Prune interaction with a competitive permeable peptide (CPP) attenuates migration of breast and neuroblastoma cells. This series of events suggests that the Nm23-H1/h-Prune protein complex regulates cancer progression and that its specific impairment could be a new therapeutic strategy in oncology. We found that CPP leads to inhibition of the AKT/mTORv and NF-kBv signaling pathways and also activates apoptosis. To obtain a proof-of-concept of our hypothesis, we used a xenograft model of prostate cancer to evaluate whether impairment of this complex using CPP results in an anti-tumoral effect. Using a mouse orthotopic model with bioluminescent imaging, we show evidences that CPP reduces prostate cancer metastases formation. In conclusion, CPP being able to impair formation of the h-Prune/Nm23-H1 complex holds promise for the treatment of prostate cancer.

The multistep process of carcinogenesis implies the accumulation of multiple molecular defects. Alteration of tumor suppressor and metastasis suppressor genes are the important steps. Increasing experimental evidence indicates that decreased expression of tumor-metastasis/suppressor genes and gene products are involved in the progression of a variety of human malignancies. In the present study, we have extended this analysis to non-small cell lung carcinomas (NSCLC). The expression and prognostic significance of the tumor suppressor gene PTEN and metastasis suppressor genes nm23-H1 and KAI-1 was evaluated in NSCLCs. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissues from 53 bronchogenic adenocarcinomas and 51 squamous cell carcinomas using monoclonal antibodies against PTEN, nm23H-1, and KAI-1 proteins. Immunohistochemical results were correlated with tumor stage, grade, lymph nodes positivity, metastasis, and patient survival. Significant co-expression of PTEN, nm23-H1 and KAI-1 was observed in NSCLC (P<.001 to .002). The immunohistochemical expression of these proteins was significantly higher in stages 1 and 2 compared with stages 3 and 4 (P=.04 for PTEN and KAI-1, P=.039 for nm23-H1). When all stages were considered together, loss of immunoreactivity for PTEN, nm23-H1 and KAI-1 was found in advanced NCSCLs (P=.015 for PTEN, P=.001 for KAI-1, P=.004 for nm23-H1), which is suggestive of co-downregulation of these proteins in the process of tumor progression. On multivariate analysis, negative staining for PTEN (P=.014), KAI-1 (P=.034), and nm23-H1 (a trend toward association for nm23-H1 reached near significance P=.08) correlated with disease-related death. Positive lymph node status was associated with negative immunostaining for PTEN (P=.007) but no correlation was observed for nm23-H1 and KAI-1. Loss of expression was linked to distant metastasis (P=.006 for PTEN, P=.002 for nm23H1, P=.001 for KAI-1). On multivariate analysis, co-downregulation of PTEN (P=.009), KAI-1 (P=.02), and nm23-H1 (P=.011) independently predicted shortened survival in NSCLC. Although NSCLC exhibits strong co-expression of PTEN, nm23-H1 and KAI-1, there is a loss of these proteins in high-stage tumors. Co-downregulation of PTEN, KAI-1, and nm23-H1 significantly correlates with distant metastasis and predicts shortened survival. Our study supports a role of these tumor suppressor and metastasis suppressor genes in the evolution and progression of NSCLC.

BACKGROUND: To analyze the prognostic significance of the genes casein kinase 2 alpha subunit (CSNK2A1), anti-apoptosis clone-11 (AAC-11), and tumor metastasis suppressor NME1 in completely resected non-small cell lung cancer (NSCLC) patients. MATERIAL/METHODS: Total RNA was extracted from 145 cases of completely resected, formalin-fixed, paraffin-embedded NSCLC tissues. mRNA expression levels of CSNK2A1, AAC-11, and NME1 were detected by real-time reverse-transcriptase polymerase chain reaction. Univariate and multivariate survival analyses were used to identify factors related to prognosis. RESULTS: A correlation between CSNK2A1 and AAC-11 mRNA expression levels (rs=0.366, p=0.000) was found. Univariate analysis showed that high expression of CSNK2A1 and AAC-11 was predictive of poor prognosis in NSCLC patients (p=0.029 and 0.044, respectively), especially when expression levels of both genes were concomitantly high (p=0.007). Multivariate Cox regression analysis showed that high expression of CSNK2A1, or concomitantly high expression of CSNK2A1 and AAC-11, are independent prognostic factors of poor survival in NSCLC patients. However, NME1 mRNA expression level did not significantly influence survival in NSCLC patients. CONCLUSIONS: This retrospective study indicates that CSNK2A1 and AAC-11, especially in combination, are useful prognosis markers in NSCLC patients after complete resection, independent of lymph node metastasis status.

The nm23 gene is a potential metastasis suppressor gene originally identified using a murine melanoma cell line. Immunohistochemical investigation on the role of nm23 gene product (nucleoside diphosphate kinase: NDPK) in squamous cell carcinomas of the oral cavity was performed and compared to clinical and histopathological factors. The stainability of NDPK did not correlate to tumour extent, differentiation and mode of invasion. However, the NDPK positive group tended to have a lower frequency of lymph node metastasis, and a better prognosis than the NDPK negative group. Consequently, nm23/NDPK is suggested to be a metastasis suppressor factor that may be useful for predicting tumour metastasis and prognosis in oral squamous cell carcinoma.

The murine nm23, a putative metastasis suppressor, has three human homologues, NM23-H1, -H2, and -H3b. Several reports have suggested a low metastatic potential for neoplasms with a high expression of NM23-H1 gene, while other studies have not shown this relationship. These apparent differences in the role of NM23 in metastasis suppression might be explained by unability to discriminate between the expression of the two genes NM23-H1 and NM23-H2. The NM23-H2 product is not related to tumor progression and metastasis suppression. Two studies on human oral squamous cell carcinoma (OSCC) have been reported, both showing the NM23 product to be a metastasis suppressor factor. However, none of these two studies distinguished NM23-H1 from NM23-H2. The aim of this study was to detect the protein expression pattern of NM23-H1 product in 24 OSCCs by immunohistochemistry in paraffin-embedded tissues using a monoclonal antibody non-cross-reactive with NM23-H2. The NM23-H1 positive group showed lower frequency of lymph node metastasis, and a better grading than the NM23-H1 negative group supporting the role of NM23-H1 as metastasis suppressor factor which may be useful for predicting tumor metastasis in OSCC.

The metastasis suppressor activity of nm23/nucleoside diphosphate (NDP) kinase was assessed using human oral squamous cell carcinoma (SCC) cell lines. When the expression of nm23/NDP kinase was compared among several SCC cell lines, nm23-H2/NDP kinase B gene product, but not nm23-HI/NDP kinase A gene product, was reduced in the metastatic cells. Transfection of nm23-H2 into the metastatic SCC cell line LMF4 caused reduction in the lung metastasis in an experimental metastasis assay. A histological analysis of the pulmonary metastatic foci revealed that although foci of the control clones were composed of anaplastic squamous cells, those of the nm23-H2-transfected clones consisted of mostly well-differentiated cells mimicking normal stratified epithelial constitution. The transfected cells were morphologically indistinguishable from the control ones in culture, but they differed from each other in that the former cells proliferated faster than the latter, became less serum dependent, and lost responsiveness to growth factors such as platelet-derived growth factor, insulin-like growth factor I, and insulin, although both clones retained sensitivity to transferrin. These results demonstrate that nm23-H2 protein does have metastasis suppressor activity for human SCC cells and suggest that this activity may be elicited by modulating growth and/or differentiation potential in response to environmental factors.

OBJECTIVE: To study the relation between the expression level of nm23-H1 protein and prognosis of oral squamous cell carcinoma (OSCC). METHODS: The expression of nm23-H1 protein in 75 cases of OSCC, 27 cases of oral mucosa, 9 cases of cervical lymph-node metastases by immunohistochemical technique (SABC), and the relation between nm23-H1 protein expression level and prognosis of OSCC was evaluated by chi-test and single-factor, multi-factor Cox proportional hazards regression model analysis. RESULTS: These immunoreactivities of nm23-H1 protein were mostly in the cytoplasm of tumor and epithelial cells. There was a significant difference of nm23-H1 protein expression level between three-year survival and seven-year survival group (P < 0.05), and there was a relation between nm23-H1 protein expression level and prognosis of OSCC in single-factor Cox analysis. But no relation was found between nm23-H1 protein expression level and prognosis in multi-factor Cox analysis. CONCLUSION: nm23-H1 protein expression level was probably not a valuable predictor of OSCC patients' prognosis. It may indirectly affect OSCC prognosis through its effect on OSCC metastasis.

OBJECTIVE: To explore the role of vascular endothelial growth factor C (VEGF-C), VEGFR-3 and nm23-H(1) in lymphatic metastasis of oral squamous cell carcinoma (OSCC). METHODS: The expression of VEGF-C, VEGFR-3 and nm23-H(1) were examined by immunohistochemical SP method. The lymphatic vessels density (LVD) in tumors was counted and analyzed with clinicopathologic parameters. RESULTS: VEGF-C, LVD and nm23-H(1) expressions were significantly different between OSCC and benign and precancerous lesions (P < 0.05). VEGF-C was correlated with lymph node metastasis and LVD (P < 0.01). nm23-H(1) was related to clinical stage, histological grade and lymphatic metastasis (P < 0.05). CONCLUSIONS: The high expressions of VEGF-C/VEGFR-3 and inactivation of nm23-H(1) may play an important role in lymphatic metastasis in OSCC.

Nm23-H1/NDPKA and Nm23-H2/NDPKB belong to a large family of NDP kinases, group of structurally and functionally closely related enzymes. The Nm23/NDPs are known to catalyse the transfer of terminal phosphates from ATP to other NTPs and dNTPs. Besides their role in the maintenance of the cells NTP pool the nm23 genes/proteins are known to have additional different biological functions, the most important being its metastasis suppressor activity. The complete picture of roles, actions and targets of nm23 genes/proteins is yet to be discovered. Our goal was to identify the downstream targets of Nm23-H2 by subjecting Nm23-H2 overexpressing CAL 27 cells (oral squamous cell carcinoma of the tongue) to microarray analysis. Using this powerful technology we identified genes, groups of genes and signalling pathways that could be clustered into several groups: apoptosis related genes, cell cycle and DNA damage, TGFbeta (transforming growth factor beta) signalling pathway and related molecules, WNT signalling pathway, differentiation and epithelial structural and related molecules, cell adhesion, metalloproteinases and their inhibitors, vesicular transport related molecules, proteasome associated, ubiquitin mediated proteolysis and several metabolic pathways. Based on these results we suggest that nm23-H2 might have an important role in oral squamous cell carcinoma which is to be confirmed by future studies.

Nm23-H1, also known as NDPK-A, was the first of a class of metastasis suppressor genes to be identified. Overexpression of Nm23-H1 in metastatic cell lines (melanoma, breast carcinoma, prostate, colon, hepatocellular, and oral squamous cell carcinoma) reduced cell motility in in vitro assays and metastatic potential in xenograft models, without a significant effect on primary tumor size. The mechanism of Nm23-H1 suppression of metastasis, however, is incompletely understood. Nm23-H1 has been reported to bind proteins, including those in small G-protein complexes, transcriptional complexes, the Map kinase, the TGF-beta signaling pathways and the cytoskeleton. Evidence supporting these associations is presented together with evidence of resultant biochemical and phenotypic consequences of association. Cumulatively, the data suggest that part of the anti-metastatic function of Nm23-H1 lies in pathways that it interrupts via binding and inactivation of proteins.

Changes in morphological features between the primary and metastatic sites in osteosarcoma and the role of nm23 protein and c-MET oncogene product have remained controversial. In addition to histological studies, we evaluated the expression of nm23, c-MET, p53, and MDM2 immunohistochemically using 25 osteosarcomas in which both primary and concordant metastatic specimens were available. Moreover, we assessed proliferative activity using the monoclonal antibody MIB-1. Among these 25 cases, 4 tumors that were osteoblastic type (16%) in the primary site had changed morphologically to MFH-like type in the metastatic site, whereas 2 MFH-like type and 1 small cell-type tumors had changed to osteoblastic type. MIB-1 LI was significantly higher in the metastatic site than in the primary site (primary, 20.02; metastatic, 26.72; P = .0209). Seventeen cases (68%) showed increased nm23 expression in the metastatic site, whereas 2 cases showed reduced expression. nm23 expression was significantly increased in the metastatic site, compared with the primary site (P = .0009). Seven cases (28%) showing negative reaction for c-MET in the primary site showed immunuoreactivity for c-MET in the metastatic site. Although there was no statistical significance, c-MET expression seemed to be more frequent in the metastatic site, compared with the primary site. Among the overall tumors, c-MET-positive tumors showed significantly higher MIB-1 LI, compared with c-MET-negative tumors (negative, 20.99; positive, 27.65; P = .0292). No significant change was observed regarding p53 and MDM2 between the primary and metastatic site. Our results suggest that rather than being a metastasis-suppressor gene, nm23 is in fact correlated with metastatic progression in osteosarcoma. Positive correlation between c-MET expression and proliferative activity also suggests that c-MET expression may play an important role in tumor progression in osteosarcomas.

Nm23-H1 and nm23-H2 are putative metastasis suppressor genes that encode nucleoside diphosphate kinase (NDPK) A and B. NDPKs form oligomers distributed between soluble and particulate fractions of cells and therefore may exert their effects as either soluble or bound proteins. To determine whether metastasis-related functions of NDPKs are mediated by their catalytic activity in membrane bound or soluble complexes, we have stably transfected highly metastatic human melanoma Line IV Cl 1 cells with wild-type and catalytically inactive (H118Y) nm23-H1 and nm23-H2 genes and assayed their metastatic potential in nude mice. Transfection with wild-type nm23-H1 and nm23-H2 genes and catalytically inactive nm23-H1 did not significantly (all p > 0.10) alter the metastatic potential of Line IV Cl 1 cells while transfection with catalytically inactive nm23-H2 significantly (p < 0.01) reduced their metastatic potential. The lack of effect of transfection with wild-type and catalytically inactive nm23-H1 suggests that neither soluble nor membrane bound NDPK A affect the metastatic potential of Line IV Cl 1 cells. The metastasis suppressive effect of catalytically inactive NDPK B overexpression suggests that competition with bound complexes containing catalytically active NDPK B inhibits metastasis of Line IV Cl 1 cells. These results imply that bound NDPK B promotes metastasis and suggest that inhibition of its function or of its binding to critical sites may be a useful approach to limit the development of metastases in human melanoma.

OBJECTIVES: To detect the nm23 expression in osteosarcoma, analyze the correlation between nm23 expression and early metastasis and to study the relation between nm23 expression with tumor differentiation and cell proliferation, as defined by Ki-67 labeling. METHODS: nm23 oncoprotein expression was measured by immunohistochemical staining using formalin fixed, paraffin embedded sections of 39 surgically resected osteosarcomas and was compared with Ki-67 expression, histological findings and early metastasis. In each sample, the grading of immunoreactivity was recorded as 0, 1+, 2+ according to the percentage of nm23 and Ki-67 labeling cells respectively. RESULTS: The cases with more than 25% positive immunoreactivity of nm23 protein were detected in 48.7% of the total cases. There was no statistical difference between nm23 expression and early metastasis, but the cases with nm23 expression appeared to have a trend to get early metastasis. nm23 expression was significantly correlated with Ki-67 expression, which was associated with differentiation of the tumor cells. CONCLUSION: The role of nm23 as a tumor metastasis suppressor in osteosarcoma is less prominent. Both nm23 and Ki-67 may help predict the possibility of early metastasis in human osteosarcomas.

Nm23-H1 is a metastasis suppressor gene whose overexpression is associated with both reduced cell motility in various cancers and increased metastatic potential in neuroblastomas, osteosarcomas, and hematological malignances. We previously reported that Nm23-H1 exerts tumor suppressor action in prostate cancer cells and that h-Prune, which is overexpressed in various tumor types, binds Nm23-H1. Moreover, blockage of the Nm23-H1/h-Prune interaction with a competitive permeable peptide (CPP) attenuates migration of breast and neuroblastoma cells. This series of events suggests that the Nm23-H1/h-Prune protein complex regulates cancer progression and that its specific impairment could be a new therapeutic strategy in oncology. We found that CPP leads to inhibition of the AKT/mTORv and NF-kBv signaling pathways and also activates apoptosis. To obtain a proof-of-concept of our hypothesis, we used a xenograft model of prostate cancer to evaluate whether impairment of this complex using CPP results in an anti-tumoral effect. Using a mouse orthotopic model with bioluminescent imaging, we show evidences that CPP reduces prostate cancer metastases formation. In conclusion, CPP being able to impair formation of the h-Prune/Nm23-H1 complex holds promise for the treatment of prostate cancer.

The aim of the study was to define the prognostic role of the metastasis suppressor gene, nm23, in 50 patients with primary ovarian cancer. Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded specimens by the primary nm23 monoclonal antibody (Novocastra, NCL-nm23 clone 37.6). Forty-two specimens (84%) showed a positive nm23 staining. The nm23 staining was more intensive in patients with normal serum CA19.9 levels, patients with nonrecurrent disease, and alive patients (p < 0.05). Nm23 expression did not correlate with common clinicopathologic parameters such as histology, grade of differentiation, International Federation of Gynecology and Obstetrics stage, and CA-125. Although the difference was not statistically significant (p = 0.11), we found that nm23 may have a favorable prognostic factor in ovarian cancer. To clarify this subject further, prospective studies on a larger population are needed.

OBJECTIVE: To evaluate the expression of cyclooxygenase (COX)-2 in ovarian serous carcinomas (OSC) and its correlation with microvessel density (MVD), nm23 expression, clinicopathologic prognostic factors and survival. METHODS: Specimens from 44 patients with OSC were evaluated by immunohistochemistry for COX-2 and nm23 expression. tumor MVD was assessed with CD34 immunostaining. The survival data of the patients were found from data files. RESULTS: 40 specimens (90.1%) showed positive COX-2 staining. Patients with high COX-2-expressed tumors had shorter overall survival, but it was not statistically significant. No correlation was found between COX-2 expression and clinicopathologic variables. There was no significant correlation between COX-2 and nm23 expression or MVD. CONCLUSIONS: COX-2 is frequently expressed in OSC. Although we could not confirm the prognostic significance of Cox-2 expression in the present cohort of OSC patients, the p value for overall survival was just slightly greater than alpha, and this result can be referred as almost significant. We considered that the limited number of cases in our study might affect the statistical analysis of our results. Further studies involving a larger number of patients are needed to clarify the prognostic significance of COX-2 expression in ovarian carcinomas.

OBJECTIVE: This study aims to investigate the impacts of CO2 pneumoperitoneum on the growth of ovarian cancer in nude mice and the expression of tumor metastasis suppressor gene (NM23-H1) and matrix metalloproteinase -2 (MMP-2) in SKOV-3 ovarian cancer cell line cancer tissue. METHOD: Forty five nude mice were used to establish ovarian cancer xenograft models by intraperitoneal injection of human ovarian cancer cell line SKOV-3. Murine xenograft models were divided into four groups: control group (only anesthetized for 0.5 h), laparotomy group (laparotomy for 0.5 h), CO2 pneumoperitoneum of 0.5 h, and CO2 pneumoperitoneum of 1 h group. Mice were killed after 12 weeks to observe intraperitoneal tumor growth and detected mRNA expression of NM23-H1 and MMP-2 in tumor tissues by RT-PCR. RESULT: Our data show that xenograft tumors grew faster in the CO2 pneumoperitoneum groups than that in control and laparotomy groups and even faster in the CO2 pneumoperitoneum of 1 h group. The mRNA expression of NM23-H1 in CO2 pneumoperitoneum groups was significantly lower than that in control group and laparotomy group (P < 0.01). Moreover, the longer duration of CO2 pneumoperitoneum negatively correlated with lower expression of NM23-H1 (P < 0.01). In contrast to NM23-H1, MMP-2 expression was significantly higher in CO2 pneumoperitoneum groups than that in the control group and laparotomy group (P < 0.01) and positively correlated with the duration of CO2 pneumoperitoneum (P < 0.01). In addition, there was a negative correlation between the expression of NM23-H1 and MMP-2 (r = -0.984, P < 0.05). CONCLUSION: The CO2 pneumoperitoneum could promote the proliferation and metastasis of human ovarian cancer in nude mice. This effect was positively correlated with the duration of CO2 pneumoperitoneum.

metastasis suppressor activities have previously been mapped to human chromosomes 17 and 11. Decreased expression of the metastasis suppressor gene NM23, which is located on chromosome 17, has been correlated with increased metastatic potential in mammary cancers. A region on human chromosome 11, from 11p11.2-p13, has been shown to suppress metastasis in rat prostatic carcinoma cells. In both cases the metastasis suppressor activity had no effect on tumorigenicity or tumor growth rate, demonstrating that the encoded activities are distinct from effects of tumor suppression. To determine whether these human chromosomes encode general or tissue-specific metastasis suppressor activities, a truncated human chromosome 17 (i.e., pter-q23) and a full-length human chromosome 11 were separately transferred into highly metastatic rat mammary and prostate cancer cell lines and tested for their ability to suppress spontaneous metastasis in vivo. These studies demonstrated that when the pter-q23 region of human chromosome 17 is retained by the microcell hybrids, the metastatic ability of both mammary and prostatic cancer cells is suppressed. In contrast, when the pter-q14 region of human chromosome 11 is retained, only the metastatic ability of prostatic cancer cells is suppressed. Additional studies demonstrated that the metastasis suppressor activity encoded by the chromosome 17 pter-q23 region is p53-independent and not due to enhanced expression of NM23 protein.

The human nm23-H1 gene product, a putative metastasis suppressor, was identified as nucleoside diphosphate kinase (NDPK) A isoform. To investigate the functional effect of nm23-H1's NDPK activity on its suppression of the components of metastatic phenotype, we transfected a human prostate carcinoma cell line, DU145, with the cDNA encoding nm23-H1 mutant protein lacking NDPK activity. The mutant nm23-H1 transfected cell lines displayed decreased invasiveness and colonization in soft agar as the wild-type nm23-H1 transfectants did when compared with the control transfected line. The results suggest that the metastasis suppressing function of nm23-H1 is independent of the NDPK enzymatic activity.

Nm23-H1 has been identified as a metastatic suppressor gene in murine melanoma cell lines. Several functions have been attributed to its activity in cancer, including a histidine kinase activity, DNA repair, and regulation of other proteins involved in metastatic formation. While in breast cancer, NM23-H1 overexpression indicates a benign status through impairing progression of disease, its function is opposite in other cancers; e.g., neuroblastoma. To further understand this dichotomy of function in cancer, we have analyzed its function in prostate cancer, in which the relationship between NM23-H1 expression and prognostic state is today controversial. In vitro, overexpression of NM23-H1 in PC3 cells inhibited their cell motility, while downregulation of NM23-H1 expression in these cells by RNA interference showed enhanced cell motility. Immunohistochemistry analysis performed on 346 prostate cancer tissue samples showed a relationship between high levels of NM23-H1 expression in the nuclei of these tumorigenic cells and elevated Gleason score, with high levels of NM23-H1 cytoplasmic staining related to metastatic stage. This retrospective survival study demonstrates that high levels of NM23-H1 expression in the cytoplasm determine recurrence of prostate-specific antigen levels only in those patients with metastatic disease. Our findings suggest a correlation between high levels of NM23-H1 protein in the cytoplasm of the cells and progression of prostate cancer to metastasis, thus definitively identifying NM23-H1 as a new negative prognostic marker in prostate cancer.

PURPOSE: Nm23 is a metastasis-suppressor protein. Decreased nm23 protein contributes to aggressiveness in many tumors. Nm23 immunoreactivity was studied in retinoblastoma and correlated with differentiation and invasiveness. METHODS: Immunohistochemistry was performed on 73 formalin-fixed, paraffin-embedded specimens of retinoblastoma. Western blot was conducted to confirm the immunohistochemical study. Prognostic features such as differentiation, invasion of choroid, optic nerve, and orbit, and metastasis were analyzed. RESULTS: Intense nm23 immunoreactivity was seen in 61% of the retinoblastomas with no invasion and faint nm23 immunoreactivity was seen in 85% of the retinoblastomas with invasion (p < 0.001). Poorly differentiated retinoblastoma showed decreased nm23 immunoreactivity compared to well-differentiated retinoblastomas (p = 0.02). An inverse correlation was observed between invasion of choroid, optic nerve, orbit, and metastasis, and nm23 immunoreactivity. Western blot assays of fresh tumor extracts confirmed the immunohistochemical findings. CONCLUSIONS: Decreased nm23 immunoreactivity was seen in poorly differentiated retinoblastomas and in retinoblastomas with invasiveness. These findings may lay the groundwork for further studies to better understand the molecular mechanisms and provide a more accurate prediction of invasion and metastasis of retinoblastoma.

OBJECTIVE: To analyze nm23-H1 protein immunohistologically in squamous cell carcinoma of the vulva. STUDY DESIGN: In 68 carcinomas, tumor type, grade, inflammation and vascular space involvement were determined. Formalin-fixed, paraffin-embedded tissue was stained for nm23-H1. Staining pattern, intensity and percentage of stained tumor cells were evaluated. Staining results were analyzed statistically univariately by the chi2 test, Kaplan-Meier survival analysis with log-rank test and receiver operator characteristic curves and multivariately by proportional hazard analysis. RESULTS: Of the tumors, 9% were negative for nm23-H1. Positive tumors showed 4 staining patterns. tumors with inhomogeneous expression of nm23-H1 tended to have shorter disease-free and overall survival. Carcinomas with nm23-H1 staining in <50% of the tumor cells more frequently developed nonlocal tumor recurrences (p= 0.018) and showed shorter disease-free survival (p = 0.017). tumors with high nm23-H1 expression were associated with patients younger than 60 years (p = 0.02). CONCLUSION: Immunohistologic staining for nm23-H1 protein shows different staining patterns and percentages of stained cells in squamous cell carcinoma of the vulva. tumors with inhomogeneous and low nm23-H1 expression are associated with a worse prognosis.

The nm23 gene has been identified as a metastasis suppressor gene. To clarify the role of nm23 as a metastasis suppressor gene in testicular seminoma, the expression of the nm23-H1 and -H2 proteins (human nucleoside-diphosphate kinase-A and -B) was immunohistochemically examined in 43 patients. Thirty-six (84%) and 21 (49%) of the 43 primary tumors were positive for the nm23-H1 and -H2 proteins, respectively. There was no significant difference in either nm23-H1 or -H2 expression between the 24 primary non-invasive tumors and the 19 primary invasive tumors, or between the 31 primary tumors without metastasis and the 12 primary tumors with metastasis. In all, and 5 of 6 metastatic tumors, the expression of nm23-H1 and -H2 proteins was observed, respectively, and the expression was not decreased in the metastatic tumors, compared to the primary tumors. In conclusion, the immunohistochemical expression of both the nm23-H1 and -H2 gene products is not associated with the metastatic status or the invasive status of testicular seminoma, and it is unlikely to be a useful non-metastatic indicator for testicular seminoma. Further studies are needed to elucidate the biological role of nm23.

Identification of multiple clinical and pathologic prognostic factors in differentiated thyroid cancer has permitted some degree of risk stratification. However, these clinical indices fail to distinguish potential intrinsic differences in tumor virulence. The nm23 gene has been identified as a potential metastasis suppressor gene that is homologous to nucleoside diphosphate kinases. Studies in human breast cancer have shown a significant inverse correlation between nm23 levels and nodal involvement/tumor recurrence. Given the possible clinical utility of a marker of metastatic potential in the management of thyroid carcinoma, we examined 34 thyroid neoplasms and a human medullary thyroid cancer (MTC) cell line (TT) for nm23 expression. Normalized nm23 expression was assessed by Northern analysis of tumor RNA. nm23 expression (tumor expression/TT cell expression, mean +/- SE) was 1.14 +/- 0.15* in MTCs (n = 5), 0.70 +/- 0.10* in follicular cancers (n = 6), 0.51 +/- 0.11 in papillary cancers (n = 19), and 0.31 +/- 0.03 in follicular adenomas (n = 4) (*p < 0.05 when compared to adenomas). Within histologic groups, we found no correlation between nm23 expression and nodal involvement of distant metastases. Our results indicate that thyroid neoplasms of different histologies express varying levels of the nm23 transcript. Although nm23 expression seems diminished in metastatic breast cancer, it appears not to be the case in metastatic thyroid cancer. The nm23 gene may therefore have different roles in the evolution and metastases of different neoplasms.

The product of Nm23 gene has been proposed as a candidate tumour metastasis suppressor protein. A strong association has been observed between reduced expression of Nm23 gene and acquisition of metastatic behaviour in some tumour cells including breast cancer and melanoma, but not in others such as colon cancer, neuroblastoma, and cervical cancer. In the present study, we examined the abundance of Nm23 mRNA in 39 thyroid tissue specimens including five multinodular goitres, one follicular adenoma, 26 papillary and three follicular carcinomas, and four anaplastic carcinomas. Nm23 was found to be expressed in all the tissue specimens. The expression was, however, variable in different stages of thyroid carcinoma. In stages I through III of differentiated thyroid carcinoma, the average level of Nm23 gene expression was comparable to that in multinodular goitres. In advanced stage of thyroid carcinoma (stage IV and anaplastic), 2-fold increase of Nm23 expression was noted. No mutations were found in the coding region of the gene. Nm23 mRNA level cannot, therefore, be used as a marker of low metastatic potential in thyroid carcinomas. The association of high level Nm23 expression with anaplastic thyroid carcinoma suggests its correlation with rapid cell proliferation.

The nm23 gene that encodes nucleoside diphosphate (NDP) kinase has been proposed as a candidate tumor metastasis suppressor in rodent experimental carcinoma models and several types of human carcinomas. The present studies were designed to investigate, by immunohistochemical analysis, whether thyroid tissues express the nm23-H1/NDP kinase and, if so, whether the nm23-H1/NDP kinase expression is correlated to tumor metastasis suppressor potential in thyroid tumors. We found that normal thyroid epithelial cells were stained weakly but homogeneously by mouse monoclonal anti-nm23-H1/NDP kinase antibody. The staining intensity of the nm23-H1/NDP kinase in benign thyroid tumors tended to be weaker than that in normal thyroid tissues. In contrast, a series of malignant thyroid tumors expressed differently the nm23-H1/NDP kinase: the intensity of the nm23-H1/NDP kinase staining was stronger in 22 of 49 malignant tumors (45%), similar in 24 (49%), and weaker in 3 (6%) compared to that in normal tissues. The comparison of the staining intensity of the nm23-H1/NDP kinase in primary lesions of tumors with lymph node metastases and those without metastases revealed no statistically significant difference. In addition, there was also no significant difference in the nm23-H1/NDP kinase staining intensity of primary and metastatic lymph node lesions of tumors with lymph node metastases. These data indicate that the nm23-H1/NDP kinase may not be a good predictive marker for tumor regional metastatic potential in malignant thyroid tumors. We conclude that in thyroid tumors the nm23-H1/NDP kinase expression may be dissociated from metastasis suppressor activity.

NME1 is a well-documented metastasis suppressor gene, with suppressor activity demonstrated across a wide spectrum of human cancers including melanoma and carcinomas of the breast, stomach and thyroid. A primary aim of the current study was to identify profiles of genes whose expression is regulated by NME1 in cell lines of melanoma and thyroid carcinoma origin. Impact of NME1 was determined by forcing its expression transiently in cell lines using a novel Ad5-based adenoviral vector (Ad5-NME1), followed 48 h later by analysis of RNA expression profiles using the U133A microarray chip. Robust NME1 expression was achieved following infection with the Ad5-NME1 adenovirus in the human metastasis-derived cell lines WM1158 (melanoma) and WRO82 (follicular thyroid carcinoma), resulting in wide-ranging effects on gene expression in both settings. A substantial proportion of the NME1-regulated genes identified in the analyses were of clear potential relevance to metastasis, such as matrix metalloproteinase-1 (MMP1), angiopoietin-2 (ANGPT2), SERPINB9 and colony stimulating factor receptor-2B (CSFR2B). Nine genes were identified (false discovery rate <0.1) that were regulated by NME1 in both the WM1158 and WRO82 cell lines, each possessing one or more such metastasis-relevant activities as stress fiber formation and focal adhesion (PPM1E, ZYX, PFN1), chemotaxis (CCR1) epithelial-mesenchymal signaling (WNT6), differentiation and morphogenesis (TBX4, ZFP36L2), and G protein modulation (GPR52 and PFN1). In addition, a number of the NME1-regulated genes were shown to be of prognostic value for distant disease-free survival and overall survival in melanoma and breast cancer. The combined expression of three NME1-regulated genes CSFR2B, MSF4A1 and SERPINB9 provided a strongly synergistic correlation with distant disease-free survival in the basal subtype of breast cancer (p<3.5e(-5), hazard ratio=0.33). Our study demonstrates that analysis of NME1-dependent gene expression is a powerful approach for identifying potential modulators of metastatic potential in multiple cancer types, which in turn may represent useful therapeutic targets. The study also highlights NME1-dependent genes as potential prognostic/diagnostic indices, which are profoundly lacking at present in melanoma.